Oral cancer is one of the most prevalent disease in developing nations.
In India, the prevalence of oral cancer is from 50 to 70% among all cancers
occurring in the body compared with 2-3% only in the UK and USA (Gupte
et al., 2001). The incidence of oral cancer is more in males than
in females. This is attributed to the indiscriminate abuse of tobacco
both in smoke and smokeless form added along with alcohol consumption.
The cancer in the oral cavity usually presents itself initially as a
non healing ulcer or an exophytic growth. When the patient suffering from
oral cancer seeks medical help, the cancer would have been in the advanced
stages, which might compromise the treatment output, leading to poor prognosis.
Presently the treatment modality for oral cancer at the advanced stages
involves extensive surgeries, chemotherapeutic agents, radiation therapy
or combination of these together, which have their own side effects. In
this scenario it would be prudent to confront this disease at it early
stages. Many researchers are extensively working upon techniques, which
would help in early detection of the disease at its premalignant and early
Presently many non invasive techniques are available like local application
of vital stains such as lugols iodine (Epstein et al., 1992), toluidine
blue (Epstein et al., 1992; Martin et al., 1998) and performing
exfoliative cytology (Ogden and Cowpe, 1989) for early diagnosis of the
disease. These techniques are very sensitive and the results depend upon
the experience of the person who performs and interprets it. On the other
hand there is a Gold standard technique called tissue biopsy which is
an invasive technique and is considered to be the confirmatory test in
the diagnosis of the lesion. Yet this technique has its own drawbacks.
It is contraindicated in certain disease conditions, it has to be performed
by an experienced dental surgeon and the right area of biopsy has to be
determined. To bridge the gap between the non invasive and invasive technique,
a new technique is needed to serve the purpose.
Autofluorescence spectroscopy is a new optical real time technique which
is under research for the early diagnosis of oral cancer. Here, the term
Autofluorescence means that the biological tissue has an inherent property
to fluoresce due to the presence of biomolecules called fluorophores when
suitably excited by ultra violet or visible (UV/VIS) light. This technique
is based upon the principle of fluorescence. One of the tissue metabolic
products is porphyrin which are formed during the hematogenic process
and also during cellular metabolism. It is well established that porhyrin
has a property to fluoresce when excited suitably (Vengadesan et al.,
1998). During carcinogenesis, there is increased cellular proliferation
and cellular metabolism associated with increased vascularity.
In this in vivo study, an attempt has been made to analyze the
fluorescent property of the tissue as it progresses from normal to malignant
MATERIALS AND METHODS
Golden Syrian hamster cheek pouch model is a well established animal
model to study DMBA induced oral carcinogenesis. Male golden Syrian hamsters
(Mesocricetus auratus, retired breeders 150-200 g) were procured
from National Institute of Nutrition, Hyderabad and maintained at the
Central Animal House, Rajah Muthiah Institute of Medical Science. The
animals were housed in plastic cages under controlled environmental conditions
with a 12 h light/dark cycle and had free access to water and standard
food. Totally, eighteen animals were taken up for the study. They were
grouped as control (n = 6), premalignant group (n = 6) and malignant group
(n = 6). A 0.5% solution of 7,12-dimethylbenz(a)anthracene (DMBA, Sigma)
in liquid paraffin oil was painted in the right buccal cheek pouch and
the left buccal cheek pouch was left untreated which acted as internal
control. The DMBA application was done two times a week for first two
weeks and then three times a week for 12 weeks. As the painting was started
there was increased inflammation with purulent discharge in the buccal
pouch. Thus time was given for the inflammation to subside and for the
animal to get conditioned to DMBA painting. For the premalignant group
animals, as soon as the clinically white patch (leukoplakia) was observed
some where around four to six weeks the application of the DMBA was stopped.
For the malignant group the DMBA was applied till a clinically apparent
tumor was observed at about the end of 14 weeks. Rajah Muthiah Medical
College, Annamalai University Institutional Animal Ethics Committee Clearance
Fluorescence measurement: The animals from the control, premalignant
and malignant group were subjected to fluorescence spectroscopy analysis
under ketaminium hydrochloride anesthesia. A fiber optic probe connected
to the Varian Fluorescent Spectrophotometer, which was, in turn, connected
to the computer, was used for the analysis. The right buccal pouch was
pulled out and the fiber optic probe was gently placed over the mucosa
of the animal in control group and over the lesion proper in premalignant
(leukoplakia) and malignant group. The tissue was excited at 405 nm and
was scanned over 420 to 750 nm range of wavelength for the fluorescence
emission by the tissue. The scan thus obtained was emission spectra.
Statistical analysis: The emission curve of the control group,
premalignant (leukoplakia) and malignant group were averaged to get individual
average emission spectra of each group. To find the statistical difference
between the average spectrum ratio parameters were introduced. With the
obtained ratio parameters through ANOVA the statistical significance was
seen between the groups.
RESULTS AND DISCUSSION
Figure 1 shows the typical autofluorescence spectra
of the normal, premalignant (leukoplakia) and oral squamous cell carcinoma.
The fluorescence intensities gradually decreased with the progression
of the scan in normal and premalignant group. There is a sharp increase
in the fluorescence intensity forming a prominent peak at around 635 nm
and a small peak at around 700 nm seen in the malignant group. No such
peaks were observed in the normal and premalignant group. In the normal,
premalignant and malignant group a small additional peak around 490 nm
was also observed. Mean ratio parameters between 490, 635 and 700 nm viz.,
I490/635, I490/700 and I635/700 were
introduced. An ANOVA test showed significant difference at p<0.05 in
the ratio value among all categories. To further identify the relationship
between groups, LSD post hoc test was performed (Table 1).
|| Average emission at 405 nm excitation
|| Results of LSD post hoc test
|*Indicates statistically significant with p<0.05
The patient suffering from initial stages of oral cancer usually does
not suffer from grave symptoms except for white or red patch with or without
ulcerations. The patient presents himself to the dental office usually
when the oral cancer is in the advanced stages, thus making it very difficult
to treat and have a very poor prognosis. To reduce the mortality rate
associated with this disease, it would be prudent to confront this disease
at its premalignant or early stages of invasion. Autofluorescence spectroscopy
is one such real time optical technique, which is under research for the
early diagnosis of cancer. This technique exploits the usage of biomolecules
called fluorophores, tissue metabolite like porphyrin which has the property
to fluoresce when excited between 400-450 nm of light (Coghlan et al.,
2000, 2001). When light of certain wavelength is absorbed by an atom or
molecule, an electron is excited to the higher energy level. When these
displaced electrons return to the original ground state it may emit a
quantum of light, which is known as fluorescence. This technique has a
many advantages compared with the non invasive procedures currently available.
(1) It can detect subtle changes in the tissue based upon tissue metabolites
(porphyrin), (2) both quantitative and qualitative analysis is possible
thus avoiding bias and (3) it is simple and very easy to perform. The
only disadvantage is that this technique cannot be used to differentiate
different pathological types of oral cancers.
In the present in vivo study, the Syrian golden hamsters buccal
cheek pouch were used for the induction of tumor by local application
of 0.5% solution of 7,12-dimethylbenz(a)anthracene in liquid paraffin.
This carcinogenesis animal model is a well established and is used in
various chemoprevention experiments due to its advantages. Induction of
tumor though various stages of occurrence such as inflammatory reaction,
hyperkeratosis, papillary stage seen in human is possible in this animal
model. Clinical distinction can be made between premalignant lesion (leukoplakic
patch) and malignant oral cancer tumor. The duration of the induction
of tumor is relatively less 14 weeks.
Bottiroli et al. (1995), Dhingra et al. (1996) and Vengadesan
et al. (1998) worked in this area and have concluded that there
is localization of porphyrins in the tumor tissue. But not many in
vivo studies are available where autofluorescent property of the oral
tissue has been studied comparing the normal, premalignant lesion (leukoplakia)
and oral cancer tissue. This has led us to take up the study to compare
the fluorescent characteristics of the tissue from normal to malignant
transformation. Porphyrin is a metabolic product in the heme biosynthesis.
It leads to the synthesis of protoporphyrin IX (PpIX), which acquires
an atom form heme. δ-Aminolevulinate synthetase, the enzyme catalyzing
the committed step in this pathway, is feedback inhibited by heme. Porphyrin
produces a red fluorescence, with peaks at 590, 630 and 690 nm when excited
in the blue spectral region between 400 and 450 nm. In this study, the
results revealed that, when the cancer tissue was excited at 405 nm there
was a prominent peak at 635 nm and another small peak at 700 nm. These
two peaks can be attributed to porphyrin fluorescence. As stated above,
there is an accumulation of the porphyrin due to the increase in the cellular
metabolism, breakdown of RBC`s and increased neovascularization (Onizawa
et al., 2002; Dhingra et al., 1996). When compared with
the normal control and premalignant group no such peak was found. Vengadesan
et al. (1998) and Onizawa et al. (2002) in their in vitro
study of malignant animal tissue found prominent porphyrin peak at 630
nm when excited at 405 nm.
The peak around 490 nm which was observed in all the three groups may
be attributed to nicotinamide adenine dinucleotide of reduced form (NADH)
(Wu et al., 2006). When mean ratio parameters between the NADH
and porphyrin peaks were introduced, statistical significance of p-value
less than 0.05 was observed between groups and within groups. In the LSD
post hoc test, it was evident that all the ratio parameter, I490/635,
I490/700 and I635/700 showed significant difference
between the normal-malignant groups and premalignant-malignant groups.
But the normal-premalignant group showed no difference. Probably some
other excitation wavelengths have to be studied to show the difference
between the normal-premalignant groups. However, from the results obtained
from the present study it is evident that autofluorescence technique shows
significant difference between the normal and diseased (malignant) tissue.
Autofluorescence spectroscopic analysis of clinically subtle lesions,
which occurs long before the apparent lesions, has to be explored. Since
this technique is noninvasive, it can help the dental surgeon to detect
early, choose the site for biopsy and to mark the boundaries of oral squamous
cell carcinoma lesion. Further in vivo studies have to be done
in order to exploit the potentiality of this technique for mass screening
at the primary clinics.
The Authors thank the University Grants Commission, New Delhi, India
to support this study under Major Research Project No. F.30-163/2004(SR).