Evaluation of PAX9 as a Novel
Biomarker in Oral Carcinogenesis
Renna Mahsoub
Supervisors: Dr Max Robinson, Dr Ralf Kist
Centre for Oral Health Research, School of Dental Sciences
Masters by Research 2011-2012
Introduction
Oral cancer is a lethal disease and a global health care problem. Within the UK, over 6000
new cases were detected in 2009 and the current statistics suggest that there has been
minimal improvement in the survival rates over the past 50 years (Cancer Research, 2009).
Oral cancer encompasses several different histological types, but the most common is
squamous cell carcinoma (OSCC), which accounts for 90% of all oral cancers (Kujan et al.,
2006a). The oral cavity can be divided into sub-sites; the most commonly affected by OSCC
are the lateral border of the tongue (Figure 1) and the floor of the mouth (Werning, 2007).
Other sub-sites; including the buccal mucosa, hard palate, and gingivae, are less commonly
affected.
Figure 1. OSCC on lateral tongue. Image
courtesy of Max Robinson.
The majority of OSCC patients are aged 65 years or older however, there has been a
recent increase in incidence in middle aged men for unknown reasons (Cancer Research,
2009). The disease is twice as common in males compared to females (Petersen, 2009).
There are several environmental risk factors for oral cancer but it is thought that tobacco
and alcohol abuse are the most common in the UK (Cancer Research, 2009)
Survival rates from oral cancer are poor with only 50% survival at 5 years (Baykul et al.,
2010). This figure had not improved despite great advances in medical knowledge and
technology. The reason for this is still unknown but many speculate it is down to factors
such as late presentation of patients with advanced disease, high loco-regional recurrence
rates, distant metastasis and the development of second primary tumours (SPTs) within the
upper-aerodigestive tract. (Baykul et al., 2010; OCIU, 2010).
Loco-regional recurrence i.e. near the original site of the cancer occurs in around 10-30%
of patients and SPTs occur in 3-4% of patients (Braakhuis et al., 2005). It was proposed that
this is due to a concept known as field cancerisation. This theory was devised by Slaughter
et al in 1953 in an article investigating changes in head and neck epithelium in oral cancer
patients. It begins with normal epithelium developing one or more genetic mutations and
changing into a patch of abnormal tissue. This accumulates more mutations and becomes a
field of atypical epithelium which subsequently progresses into carcinoma (Tabor et al.,
2001). As the model progresses, the surface area affected by mutations is increasing
resulting in a greater risk of local recurrence or SPTs within the upper aero-digestive tract.
Potentially Malignant Disorders
Many OSCCs are preceded by a potentially malignant disorder (PMD) (Reibel, 2003)
which may be noticed by the patient or detected by a professional. These lesions vary
clinically in terms of size, shape and colour (Mehrotra and Gupta, 2011). These lesions are
usually pain-free and can go unnoticed by the patient thus making late presentation of
disease a common feature in oral cancer patients (van der Waal, 2009). The progression of a
PMD to cancer is unpredictable, it may remain as a non-invasive patch, it may regress or it
may progress to a malignant neoplasm (Mishra, 2012). There is a lack of evidence of how
each individual lesion should be treated: it can be monitored or removed using a laser
(termed laser excision) depending on patient and doctor preference. Unknown progression
poses a variety of risks and problems for both the patient and doctor. For a disorder which is
destined to become invasive, early knowledge of this progression would help deliver a more
targeted approach to treatment and as explained previously, early diagnosis decreases
morbidity and mortality from cancer (Mishra, 2012).
Biomarkers and Genetic Changes in Oral Cancer
A biomarker identified in the initial biopsy of a PMD which predicts the succession of the
lesion, could revolutionise management of these areas (Arellano-Garcia et al., 2010).
Currently, there is no robust biomarker of this description for OSCC but many possibilities
have been documented and tested, the most prominent being EGFR overexpression
(Grandis and Tweardy, 1993). Dyplasia grade can give an indication of progression to
malignancy but is far from accurate (Kujan et al., 2006b; Kujan et al., 2007). The
characteristics of a model biomarker are described in Table 1.
Attributes of an Ideal Biomarker
Measured objectively
Measured in small samples
Altered in high-risk tissues compared to normal
tissues
Altered in early carcinogenesis
Table 1. Features required for the ideal biomarker.
Adapted from (Wu et al., 2010)
Molecular changes in the tissue, for example, loss of heterozygosity (LOH) have been
suggested as possible biomarkers for OSCC (Zhang and Rosin, 2001). LOH describes a loss of
DNA from a chromosome and can be used to detect areas at risk of cancerous changes. It
has been used previously in head and neck cancers to show that the chromosomes coding
for the tumour suppressor genes p53 and p16 are affected (Forastiere et al., 2001). These
genes are known to be heavily implicated in head and neck tumorigenesis. Another
potential novel biomarker for OSCC is a key transcriptional regulator called PAX9.
PAX
The PAired-boX (PAX) family of genes are essential transcription factors for controlling
tissue differentiation and organogenesis during development in embryos (Li and Eccles,
2012). They control these pathways by encoding for transcription factors which will
influence the expression of downstream developmental genes (Muratovska et al., 2003).
PAX genes are also necessary for mediating cell proliferation, preventing apoptosis and
stem-cell self-renewal (Robson et al., 2006). Therefore it is conceivable that dysregulation of
the PAX family may be a key step in carcinogenesis (Li and Eccles, 2012).
The PAX family consists of 9 different genes (PAX1-PAX9) divided into 4 subgroups
according to their structure as shown in Figure 2. The structure of the gene directly relates
to the tissues they influence during embryonic differentiation and their effect on cancer
development (Muratovska et al., 2003).
Paired Box
Octapeptide
Homeodomain
PAX1
Group I
PAX9
Improved
Prognosis
PAX4
Group IV
PAX6
PAX2
Group II
PAX5
PAX8
Poor
Prognosis
PAX3
Group III
PAX7
Figure 2. Basic structure and sub-classifications of the PAX gene family.
The octapeptide may be present or absent and the homeodomain may be present, absent or
truncated as seen in group II. Subgroups I and IV are associated with a more positive
outcome and decreased malignancy of the cancer whereas groups II and III are the opposite
and associated with a worse outcome. (Adapted from (Muratovska et al., 2003; Robson et al.,
2006))
PAX9
PAX9 is the main PAX gene responsible for differentiation of the oral structures.
Expression of Pax9 (mouse orthologue of PAX9) in the mouse embryo has been studied in
depth and shows it is involved in endodermal epithelium formation including oral, tongue
and oesophageal stratified squamous epithelium and mesenchymal craniofacial and tooth
development (Chi and Epstein, 2006). Genetic defects in PAX9 in humans leads to non-
syndromic congenitally missing teeth and in mice will also lead to craniofacial and skeletal
abnormalities (Lee et al., 2008).
PAX genes are involved in formation of a wide variety of cancers and PAX9 is implicated in
oral and oesophageal cancer although the details of this process are unclear (Gerber et al.,
2002). PAX9 is associated with a more positive prognosis for cancer and the loss of
expression may suggest an increased risk of malignancy compared to a lesion expressing
PAX9 (Robson et al., 2006). This theory is supported by Gerber et al when in 2002 they
demonstrated that PAX9 expression is reduced in dysplasia and absent in a number of
oesophageal cancer specimens. They included at least one area of histologically normal
appearing epithelium in every biopsy to act as an internal control. This ‘normal’ tissue
retained PAX9 expression. Gerber et al concluded that the PAX9 gene plays a role in the
malignant transformation of cells.
PAX9 expression is found in the epithelium of oral mucosa biopsies and can be assessed
using immunohistochemistry (IHC).
Aims and Objectives
Aim
Assess the efficacy of PAX9 gene expression in predicting the malignant transformation of
potentially malignant disorders (PMD) into oral squamous cell carcinoma (OSCC).
Objectives
Qualitatively determine the PAX9 expression pattern in normal oral tissue, dysplasia and
OSCCs
Quantitatively determine the PAX9 expression pattern in normal oral tissue and dysplasia
Correlate PAX9 expression with patient demographics, dysplasia grade and clinical outcome
Methods and Materials
Case Selection
In total, 199 normal, dysplastic and cancerous oral biopsies were identified and
subdivided by clinical outcome as shown in Table 2.
Group Name
Number
Normal Tissue
24 cases
No Adverse Outcome
30 cases
Recurrence
15 cases
New Lesion
11 cases
Malignant Transformation
22 cases
OSCC
97 cases
Table 2. Final groups and case numbers
Each biopsy section was stained for PAX9 expression using immunohistochemistry.
Controls
The normal tissue sections were used as controls for IHC staining. Once batches of PMD
slides were stained, they could be compared to normal sections to check that the protocol
had worked. If staining was weak or the pattern unexpected, there was often histologically
normal appearing epithelium adjacent to a PMD which could be used as an internal control.
This epithelium appears disease-free when examined under a microscope.
Image Analysis
The PMD and normal tissue sections were subject to computer image analysis for
positively stained nuclei and strongly positively stained nuclei. A standardised protocol for
computer analysis is not currently available; consequently a novel method was developed.
Previous investigations suggest that the cancer specimens do not exhibit high PAX9
expression and areas of epithelium can be difficult to isolate thus computer analysis was not
carried out for the OSCCs, these were analysed descriptively. This was completed using the
Allred scoring system already in use for IHC EGFR staining in breast cancer (Allred et al.,
1998). The normal and dysplastic biopsies were also analysed descriptively.
Ethical approval was obtained from the National Research Ethics Service (NRES)
Committee North East - Sunderland (REC reference: 11/NE/0118).
Results
PAX9 Staining
Examples of the PAX9 staining after computer analysis are shown in Figure 3, 4, 5 and 6.
PAX9-positive nuclei are stained brown and the remaining tissue is counterstained blue. The
Aperio immunohistochemistry image analysis system highlights strongly stained cells in red,
followed by orange then yellow for decreasing intensity. Negative nuclei are highlighted in
blue.
Figure 3. Aperio screenshot of
normal tissue
Figure 4. Aperio screenshot of
histologically normal tissue
Figure 1. Aperio screenshot of
histologically normal tissue
Figure 1. Aperio screenshot of
normal tissue
Figure 5. Aperio screenshot of
low grade dysplasia
Figure 6. Aperio screenshot of
high grade dysplasia
PAX9 Expression Analysis
For the computer analysis, expression was defined by two parameters: percentage
positive stained nuclei (PN) and percentage strongly positive stained nuclei (PN+). For the
Allred analysis, expression was defined by a combined score for intensity and proportion of
nuclei stained.
No significant relationship was found between PAX9 expression and patient age, sex or
lesion sub-site for both PN and PN+. Significance was found between dysplasia grade and
clinical outcome.
Figure 7 demonstrates that PAX9 expression falls in all grades of dysplasia including
histologically normal appearing epithelium adjacent to PMD.
Figure 7. PN and PN+ means for WHO dysplasia grading system
80
70
62.6
PN
PN+
60
50
36.7
31.2
% 40
30
23.6
25.8
22.2
20
6.3
10
4.1
2.8
3.8
2.8
0.0
0
Normal
Histologically
Normal
Mild
Moderate
Severe
CIS
All groups for PN (p<0.03) and PN+ (p<0.045) are significantly lowered compared to normal
tissue.
Histologically normal tissue was significantly higher in PN for mild dysplasia (p=0.048),
moderate dysplasia (p=0.06) and CIS (p=0.042)
No other realtionships were found.
Figure 8 shows the relationship between PAX9 expression and clinical outcome.
Figure 8. PN and PN+ means for all clinical outcome groups
80
62.6
PN
70
60
PN+
45.8
50
% 40
30
25.7
23.3
14.8
14.3
20
10
1.2
3.3
0.1
9.1
0
Normal
No Adverse
Outcome
Recurrence
New Lesion
Transformed
PN (p<0.025) and PN+ (p<0.001) are significantly lowered in disease compared to normal
tissue. The transformed group expression was also statistically elevated compared to all the
other outcome groups in PN (p<0.02) and all groups apart from new lesion in PN+ (p<0.01).
Using the Allred scores to include the T1 and T2 cancers, expression follows the pattern
as shown in Figure 9.
Figure 9. Allred score means for all clinical outcome groups including OSCC
8
7.2
7
5.7
Allred Score
6
4.7
5
4.0
4.1
3.3
4
3
2
1
0
Normal
No Adverse
Outcome
Recurrence
New Lesion Transformed T1, T2 OSCC
All groups including the OSCC group have a significantly decreased PAX9 expression
compared to normal tissue (p<). All groups apart from new lesion display a significantly
lowered PAX9 expression compared to the transformed group (p<).
Kaplan-Meier survival curves were created from the above data. Interestingly, they
demonstrate that 90% of dysplasias with a relatively higher proportion of PAX9-positive
nuclei transformed into oral cancer within 80 months of diagnosis (Figure 10).
Figure 20. Kaplan-Meier Curve for Malignant Transformation in
Dysplasia Patients: Aperio PN in Thirds.
The curve shows 90% of dysplastic lesions with PN values of >40%
transformed into cancer compared to 41% of lesions with PN values of
21-40% and 37% of patients with 0-20% expression up to 76 months
after diagnosis
Discussion
There have been some interesting results produced by this investigation. A new
quantitative method for IHC image analysis has been very successful. With optimisation of
the protocol, this could prove to be a very powerful analysis tool.
The PAX9 expression throughout cancer formation is not as straight-forward as expected.
Within this patient cohort, PAX9 expression is decreased in dysplastic potentially malignant
disorders compared to disease-free tissue including in histologically normal appearing
epithelium. This implies PAX9 expression may be used as a screening tool to identify
epithelium in very early stages of ‘invisible’ carcinogenesis. In a clinical setting, the PAX9
expression pattern could be used by the pathologist to determine if a clear margin has been
left by the surgeon after laser excision of the lesion. Alternatively, if a patient presents with
a PMD, biopsies from nearby sub-sites can be taken to determine the spread of disease.
From here, the relatively higher PAX9 expression biopsies were more likely to transform
into cancer suggesting PAX9 moves from a potential tumour-suppressor role as previously
suggested to a tumour-promoter role. A transient upregulation of PAX9 may be causally
involved in the transformation to cancer. If this is the case, patients may need to have the
PAX9 expression in their dysplastic lesion reviewed on a regular basis to identify this
increase before expression falls again in cancer.
Conclusion
In conclusion, PAX9 is a promising prognostic marker which meets all of the criteria for an
ideal biomarker. At present PAX9 expression can act as an aid to current prognostic tests
but cannot replace these until more is known about the mechanisms of action. Further
investigations using a larger patient cohort with standardised treatments will be required.
WORDS: 1946
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