Role of brush biopsy and DNA cytometry for prevention, diagnosis, therapy, and followup care of oral cancer.

Hindawi Publishing Corporation
Journal of Oncology
Volume 2011, Article ID 875959, 7 pages
doi:10.1155/2011/875959
Review Article
Role of Brush Biopsy and DNA Cytometry for Prevention,
Diagnosis, Therapy, and Followup Care of Oral Cancer
Alfred Bo¨cking,1 Christoph Sproll,2 Nikolas Sto¨cklein,3 Christian Naujoks,2
Rita Depprich,2 Norbert R. Ku¨bler,2 and Jo¨rg Handschel2
1 Institute of Cytopathology, Heinrich-Heine-University, Moorenstraβe 5, 40225 Du¨sseldorf, Germany
2Department for Cranio- and Maxillofacial Surgery, Heinrich-Heine-University, Moorenstraβe 5, 40225 Du¨sseldorf, Germany
3Department of General, Visceral, and Pediatric Surgery, University Hospital Du¨sseldorf, Heinrich-Heine-University,
40225 Du¨sseldorf, Germany
Correspondence should be addressed to Alfred Bo¨cking, alfred.boecking@uni-duesseldorf.de
Received 30 September 2010; Accepted 21 November 2010
Academic Editor: Pankaj Chaturvedi
Copyright © 2011 Alfred Bo¨cking et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Late diagnosis resulting in late treatment and locoregional failure after surgery are the main causes of death in patients with oral
squamous cell carcinomas (SCCs). Actually, exfoliative cytology is increasingly used for early detection of oral cancer and has
been the subject of intense research over the last five years. Significant advances have been made both in relation to screening and
evaluation of precursor lesions. As this noninvasive procedure is well tolerated by patients, more lesions may be screened and thus
more oral cancers may be found in early, curable stages. Moreover, the additional use of DNA image cytometry is a reasonable tool
for the assessment of the resection margins of SCC. DNA image cytometry could help to find the appropriate treatment option
for the patients. Finally, diagnostic DNA image cytometry is an accurate method and has internationally been standardized. In
conclusion, DNA image cytometry has increasing impact on the prevention, diagnostic, and therapeutical considerations in head
and neck SCC.
1. Introduction
Patients with squamous cell carcinomas of the oral cavity
have a fair prognosis with an overall 5-year survival rate of
about 45% [1]. Unfortunately, this figure has not substan-
tially improved during the past 30 years [2]. Late diagnosis
resulting in late treatment and locoregional failure after
surgery or even after combined surgery and radiotherapy are
the main causes of death in patients with oral squamous cell
carcinomas.
These days, an alternative method for the examination
of oral lesions is exfoliative cytology. It is based on the
technique of Papanicolaou, which is accepted worldwide, as a
successful method in order to screen for epithelial dysplasias
in situ or invasive carcinomas of the uteri cervix. Currently,
exfoliative cytology is increasingly used for early detection
of oral cancer and has been the subject of intense research
over the last five years [3, 4]. Significant advances have been
made both in relation to screening and in the evaluation
of precursor lesions [5–11]. Although mucosal biopsy is
still regarded as the gold standard for definitive oral cancer
diagnosis, exfoliative cytology is a valuable tool for the
noninvasive evaluation of a range of potentially preneoplastic
oral mucosa lesions, like leuko-/erythroplakias and lichen
ruber. The cytometric detection of DNA aneuploidy in
exfoliated suspicious respectively dysplastic cells, qualifies
these as malignant, up to two years earlier than cytology or
histology alone [12, 13].
2. Prevention
2.1. Precursor Lesions of Oral Cancer. Oral carcinogenesis
proceeds through a stepwise accumulation of (cyto)genetic
changes over time. Because the oral cavity is easy to
examine and risk factors for oral cancer are known, there
is great opportunity to improve patient outcomes through
diagnosis and treatment of premalignant lesions before the

2 Journal of Oncology
development of invasive oral carcinoma [14]. In contrast to
the oral premalignant conditions, oral premalignant lesions
are morphologically abnormal solitary or multiple areas of
mucosa that are typically white, red, speckled or verrucous
in appearance. The WHO classification [15] combines
leukoplakia and erythroplakia into “precursor lesions,” with
a 6.8% estimated rate of transformation of oral leukoplakias
to cancer. It identifies proliferative verrucous leukoplakia as
a separate high risk lesion with minimal cytological atypia.
Oral lichen planus, a chronic inflammatory condition, also
is associated with an increased risk of cancer development of
about 3% [16, 17].
2.2. Indications for Brush Biopsy. Screening for oral cancer
and its precursor lesions may be performed by dentists, den-
tal surgeons, and other health care professionals. Exfoliative
cytology, taking brush biopsies, is advocated for evaluation
of macroscopically suspicious lesions of the oral mucosa that
are detected clinically by screening. This may be followed by
a mucosal scalpel biopsy. Yet, exfoliative cytology may replace
tissue biopsy in lesions that are clinically not obviously
suspicious for malignancy but nevertheless need surveillance.
As tissue biopsy is associated with lower compliance by
patients as compared to brush biopsy, this noninvasive
approach may lead to a higher number of investigated sus-
picious oral lesions and thus to an increased rate of curable
cancers, identified in early stages.
3. Sampling of Cells
Collection devices suitable to obtain cells from the super-
ficial and intermediate layers of the oral mucosa may be
conventional brushes, as used for endocervical sampling
by gynecologists, such as the CytoBrush and Orca Brush
(Figure 1). The brush is rotated under slight pressure several
times on the suspicious lesion. Cells are then immediately
smeared on glass slides and fixed with alcoholic spray. Signs
of dysplasia and malignancy will also be detected cytologi-
cally in the upper layers of the squamous epithelium due to
the principle of migration of cells from basal to superficial
layers. The degree of nuclear abnormality in the surface layers
reflects the degree of disturbance of maturation of the whole
thickness of the epithelium. Thus, transepithelial sampling
is not required to diagnose dysplasia and malignancy of the
squamous epithelium on brush biopsies.
4. Assessment of Dysplasia
There are several schemes for grading dysplasia in biopsies
of oral precursor lesions. The WHO classification provides
a five-step system: hyperplasia, mild, moderate, and severe
dysplasia followed by carcinoma in situ [15]. Squamous
cell carcinoma will develop from antecedent dysplastic oral
mucosal lesions if an early diagnosis has not been made
and treatment given. Early diagnosis within stages Tis or T1
correspond to a vastly improved 5-year survival rate when
compared with more advanced lesions (96,7%) [17, 18]. It is
the task of a cytopathologist to identify nuclear abnormalities
Figure 1: Brush biopsy from an oral verrucous leukoplakia.
in squamous cells collected to predict the histological grade
of dysplasia. The diagnostic criteria used are well known and
similar to those in cervical exfoliative cytology according to
Papanicolaou [4]. Although the degree of dysplasia can be
predicted on cytological samples (Figure 2), tissue biopsy is
usually performed when dysplasia is detected cytologically,
to confirm its grade and exclude the presence of invasion.
The latter cannot be reliably assessed by exfoliative cytology
alone. However, poor interobserver reproducibility in the
histological assessment of oral premalignant lesions is well
described [8].
5. Diagnostic Impact
5.1. Spectrum of Cytological Diagnoses. Apart from squa-
mous cell carcinoma and its precursors (dysplasias), further
neoplasias can be specifically diagnosed cytologically (e.g.,
naevuscell naevi, malignant melanomas, basalcell carcino-
mas, and malignant lymphomas). Moreover, a spectrum of
non-neoplastic diseases can be differentiated using exfolia-
tive cytology, for example, pemphigus vulgaris, Candida,
herpes simplex, and HPV infections [4].
5.2. Diagnostic Accuracy of Cytology. Cytopathologic evalua-
tion of oral brush biopsies from leukoplakias and erythro-
plakias as a single method yields sensitivities for the detec-
tion of oral cancer slightly below those of histopathologic
evaluation of scalpel biopsies, reported to be 97,5% [19].
Remmerbach et al. [5, 20] documented 91,3% and 94,6%
sensitivity of oral brush biopsy and Maraki et al. [12] even
100% for the detection of oral cancer, including the in situ
stage. Respective specificities were 99,5%, 95,1%, and 97,4%.
Moreover, 24,1% of cancers were identified in early, curable
stages Tis and T1 [20].
It is supported by an increasing number of data that oral
cytology is also a valuable technique for the assessment of
oral premalignant lesions [3, 12, 21]. Exfoliative cytology has
been shown to detect dysplasia in suspicious oral lesions with
high sensitivity and specificity by several groups [20].
Up to 5–14% of oral brush biopsies may yield to equiv-
ocal cytological diagnoses [5, 20]. Underlying diagnoses are
mild, moderate, or marked dysplasia, abnormal regenerating

Journal of Oncology 3
(a)
(b)
(c)
Figure 2: Normal (a), dysplastic (b), and malignant (c) oral
squamous cells from brush biopsy, Papanicolaou stained, 630x.
squamous epithelium, or just scarcity of abnormal cells. In
these cases, ancillary methods are desirable that, neverthe-
less, allow more definite, correct cytological diagnoses.
Meanwhile, use of auxiliary methods such as DNA image
cytometry, AgNOR analysis, and multimodal cell analysis has
been shown to significantly increase diagnostic accuracy of
oral cytology [12, 13, 20, 22, 23]. These methods are only
applied on those samples that reveal doubtful or suspicious
(dysplastic) cells, on neither cytologically normal nor frankly
malignant ones.
6. Auxiliary Cytometry
DNA image cytometry is based on microdensitometric DNA
measurements of several hundred atypical cells in routine
2.16 c 2.15 c 2.14 c 2.12 c 2.12 c 2.12 c
6.9 c 6.31 c 5.89 c 5.65 c 5.58 c
3.38 c 5.15 c 4.72 c 4.71 c 4.7 c
4.7 c 4.69 c 4.47 c 4.32 c 4.25 c
Reference cells-normal epithelial
Analysis cells
Moticyte-DNA-manual report
Figure 3: Six nuclei from normal and Feulgen-stained oral squa-
mous cells with regular DNA content (green) as internal reference
(around 2,0 c) and 15 from atypical cells with abnormal DNA
content (red) between 4.25 c 6.90 c, indicative of malignancy.
cytological specimens (Figure 3). It aims to distinguish true
prospectively malignant lesions (dysplasias) from micro-
scopically atypical or otherwise doubtful ones. The biological
basis of this ancillary method is chromosomal aneuploidy
which is an accepted marker of malignant transformation
of cells if it occurs clonally [24]. The cytometric DNA
aneuploidy (Figure 4) utilizes the fact that gains or losses
of chromosomes or their parts result in a plus or minus
of more than 10% of nuclear DNA mass in a growing
cell population (stemline aneuploidy) or if extremely high
nuclear DNA values >9 c (single-cell aneuploidy) occur [24].
DNA stemlines (modal values) outside 2 c, 4 c, or 8 c ± 10%
are regarded as abnormal (resp., aneuploid, Figure 4) [23,
25]. Measurements may be performed on previously stained
slides after destaining and Feulgen restaining. Morphologi-
cally suspicious cells are interactively selected on a monitor,
and internal calibration is performed with normal (e.g.,
intermediate squamous) cells (Figure 3). The method has
been internationally standardized and is applicable to many
different epithelial dysplasias [24–26]. After enzymatic cell
separation, DNA image cytometry (ICM) can also be applied
on formalin-fixed and paraffin-embedded tissues, that is on
all histologic routine specimens like biopsies and resected
tissues [27]. Thus, even histologic diagnoses of dysplasias can
be subjected to DNA cytometry to predict their prospective
behavior.
Remmerbach et al. [5] reported a frequency of 13.9%
doubtful or suspicious oral cytological diagnoses due to

4 Journal of Oncology
different grades of squamous dysplasia or abnormal regen-
erating epithelium. Applying DNA aneuploidy as a marker
for prospective malignancy on identical slides, they could
improve diagnostic sensitivity of cytology for the detection
of oral cancer from 91.3% to 97.8% and specificity from
95.1% to 100%. Thus 29.4% of oral cancers that clinically
appeared as leukoplakias or erythroplakias were detected
in stages Tis or T1. In a similar study Maraki et al. [12]
described a sensitivity of 100% and specificity of 97.4% for
the combined cytological and DNA cytometric evaluation
of oral leukoplakias and erythroplakias. 8.1% of their cyto-
logical diagnoses had been equivocal. DNA-ICM was only
applied if one of the above-mentioned diagnoses (mainly
dysplasias) had occurred. Seven cases in which combined
cytological/DNA cytometric diagnosis of early oral cancer
was achieved up to two and half years before definitive biopsy
diagnosis have been published [12, 13]. Thus DNA-ICM
may help to predict the prospective behavior of cytologically
suspicious lesions, as the positive predictive value of DNA
aneuploid findings was reported to be 100% and the negative
value 98.1% [13, 20].
Another auxiliary method that allows assessment of
potential malignancy of dysplastic or regenerating cells is
AgNOR analysis. AgNORs represent silver-stainable nucle-
olar organizer regions (Figure 5). Their number and size
are related to protein synthesis. Remmerbach et al. [13, 23]
showed that counting the number of silver nitrate-stained
nucleolar organizer regions (AgNORs) in about 100 atypical
squamous cells allows 100% sensitivity and specificity of oral
cancer detection on brush biopsies.
Both methods, DNA-ICM and AgNOR analysis, may
even be performed sequentially on identical cells (Figure 5).
This type of multimodal cell analysis is especially useful
if only few atypical cells are available [23]. Thus, AgNOR
analysis can be combined with DNA-ICM if the latter does
not yield an unequivocal result.
7. Role in Therapy
Treatment method of choice in patients with squamous cell
carcinomas of the head and neck area is still surgical resection
of the tumor and dissection of the regional lymph nodes.
Although options for repair and restoration (e.g., free flaps)
of skin and bone defects after primary surgery have improved
significantly in the last decades, patients with squamous cell
carcinomas of the oral cavity have only a fair prognosis
with an overall 5-year survival rate of about 45% [1]. This
figure has not substantially improved during the past 30
years [2, 28, 29]. Locoregional failure after surgery or even
after combined surgery and radiotherapy is the main cause
of death in patients with squamous cell carcinomas of the
mandibular region and the maxilla. The main principle in
tumor surgery is the effort to achieve tumor-free resection
margins.
Several authors have evaluated the relationship between
locoregional recurrence of the tumor and the status of
the resection margins [30, 31]. The prevalence of tumoral
infiltration at the resection margins varies from 3.5% to
0
20
C
ou
n
t
0 2 4 6 8 1210 14 16 18
(C)
Abnormal (A)
Normal epithelial (R)
DNA-histogram (C) for 13550-09
Figure 4: DNA histogram of in-situ oral carcinomas cells, revealing
abnormal stemlines (red) at 3.5 c and 6.5 c, and values up to 17 c
(DNA aneuploidy), indicative of malignancy. Normal epithelial cells
(green bars) at 2 c.
Figure 5: Illustration of three sequential stainings of identical
oral cancer cells as performed in multimodal cell analysis [27]:
Papanicolaou, Feulgen for DNA analysis, and silver nitrate for
AgNOR analysis. Black dots represent AgNORs.
60% [30] and is usually an indicator for additional excision,
postoperative irradiation, and strict followup [32]. The
recurrence rate in patients with positive surgical margins
treated only by surgery ranges from 36% [31] to 64% [30],
and when postoperative radiotherapy is used, the recurrence
rate decreases to 31% [30]. Due to the fact that it can be
difficult to distinguish between squamous cell carcinomas
and other lesions of the oral mucosa using only haematoxylin
and eosin-stained sections [33] the resection margins are
routinely examined by immunohistology. Nevertheless, the
histological diagnoses of oral mucosa lesions fail sometimes
[34, 35]. These days, an alternative method for the examina-
tion of oral lesions is exfoliative cytology. It is based on the
technique of Papanicolaou, which is accepted worldwide, as a
successful method in order to screen for epithelial dysplasias
in situ or invasive carcinomas of the uteri cervix. Moreover,

Journal of Oncology 5
DNA image cytometry has been introduced for diagnosis of
malignant transformation of squamous epithelial cells as an
adjuvant tool to the cytological examination [20, 36]. This
is used to detect the cytometric equivalent of chromosomal
or DNA aneuploidy [37], which is accepted as a marker
for the neoplastic transformation of cells. DNA image
cytometry has been introduced as an adjuvant tool for the
detection of these cell transformations in oral mucosa [20,
36]. The detection of DNA aneuploidy has been described
as a diagnostic aid for the identification of prospective
malignancy in various organs for example in dysplasias of
the uterine cervix [38], suspicious cystic lesions of the neck,
[39] or bile duct brushings [40]. The positive predictive
value of DNA aneuploidy for the subsequent deletion of
histologically confirmed cancer was 100% in cells of these
tissues. In another study, the additional value of DNA
image cytometry regarding the occurrence of a locoregional
relapse was assessed [27]. In this study adjuvant use of
DNA image cytometry showed a high positive predictive
value of 87.5% with respect to the local recurrence of head
and neck squamous cell carcinomas. Recently, Brandizzi and
coworkers reported a ploidy analysis in oral squamous cell
carcinomas using methodologic adjustments to improve the
accuracy of the measurements of aggressiveness of prognostic
value. Several indices of aggressiveness were analyzed in
relation to the clinical-pathologic data and evolution of the
patients. Two indices had a prognostic value of the degree of
aggressiveness of oral SCC [41].
Taking into account that the diagnosis of tumor infiltra-
tion in the resection margins has often serious consequences
(followup resection and/or postoperative irradiation), the
presence of aneuploid cells could also change the treatment.
However, it is unclear if these aneuploid cells cause the
locoregional tumor relapse. Unfortunately, up to date no
studies exist which confirm this. Thus, it has to be investi-
gated in a consecutive clinical trial, whether the additional or
modified treatment leads to a longer relapse-free period.
In conclusion, the additional use of DNA image cytom-
etry is a reasonable tool for the assessment of the resection
margins of SCCs. DNA image cytometry could help to find
the appropriate treatment option for the patients and thus
might improve their prognosis.
8. Followup Care
Local recurrences of oral cancer after operation are frequent
events, more often following R 1/2—but even after R0—
resections [27]. Exfoliative cytology allows the non-invasive
evaluation of macroscopically suspicious mucosal lesions
that may appear after resection. As brush biopsies are better
tolerated by patients than scalpel biopsies, they may be
performed more often. Thus, recurrences may be identified
earlier.
9. Conclusion
DNA image cytometry has tremendous impact on early
diagnosis and therapeutical considerations in head and neck
squamous cell cancer. While oral lesions that macroscopically
are urgently suspicious for cancer shall be submitted to
scalpel biopsy and histologic evaluation, the majority of
facultatively precancerous lesions, such as leuko- and ery-
throplakias or even persistent lichen planus lesions, may be
assessed by brush biopsy and cytology. As this non-invasive
procedure is well tolerated by patients, more lesions may be
screened and thus more oral cancers may be found in early,
curable stages. Oral brush biopsies can easily be performed
by dentists, dental surgeons, and general practitioners. While
sensitivity of exfoliative cytology alone is about 4% less than
bioptic histology, the combination of the latter with DNA
image cytometry reaches the same diagnostic accuracy as the
former. As clonal chromosomal aneuploidy and DNA aneu-
ploidy mostly precede cytological and histological evidence
of malignancy in the squamous epithelium, its detection
allows the diagnosis of oral squamous cell carcinomas up
to two years earlier. Moreover, the additional use of DNA
image cytometry is a reasonable tool for the assessment
of the resection margins of squamous cell carcinomas.
DNA image cytometry could help to find the appropriate
treatment option for the patients and thus might improve
their prognosis.
Finally, diagnostic DNA image cytometry is an accurate
method and has internationally been standardized. Actually,
it is paid by the German health insurances.
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