CLINICAL REVIEW

David W. Eisele, MD, Section Editor

Adjunctive diagnostic techniques for oral lesions of unknown malignant potential:

Systematic review with meta-analysis
Colin Fuller, MD, MS,1* Ryan Camilon, BA,1 Shaun Nguyen, MD, MA,1 Jon Jennings, MD,2 Terry Day, MD,1 M. Boyd Gillespie, MD, MSc,1
1

Department of OtolaryngologyHead and Neck Surgery, Medical University of South Carolina, Charleston, South Carolina, 2Department of Emergency Medicine, Medical University
of South Carolina, Charleston, South Carolina.

Accepted 1 March 2014

Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23667

ABSTRACT: Background. The purpose of this study was to critically

review the published evidence concerning adjunctive diagnostic techniques in the diagnosis of oral lesions of unknown malignant potential.
Methods. We conducted a systematic literature review with metaanalysis using PubMed to search for articles published from June
1993 through June 2013 to identify prospective studies evaluating
any diagnostic method, with tissue biopsy confirmation, in clinically
evident oral lesions of unknown malignant potential. Aggregate
weighted totals and SEs for true, false-positive, false-negative, and
inadequate results were calculated and compared among
subgroups.

INTRODUCTION
Oral cancer is the eighth most common cancer worldwide.1 In the United States alone, there were an estimated
27,450 new cases of oral cancer in 2013, and it caused an
estimated 5,490 deaths.2 Although not among the most
common cancers in the United States, oral cancer is associated with a high degree of morbidity and mortality,
which varies widely with staging. It has been demonstrated that early detection is crucial to optimal outcomes.
As of 2009, pooled five-year relative survival for stage
III and IV disease for oral and oropharyngeal cancer was
estimated at 59.2%, whereas that of stage I and II disease
was 82.7%.3 In certain parts of the world, oral cancer
incidence is much higher. In south-central Asia, cancer of
the oral cavity is among the three most common types of
cancer.1 Despite the need for earlier diagnosis of oral cancer, in the United States, only 33% of oral and oropharyngeal cancer was diagnosed at an early, localized stage
from 2006 to 2010.4 Because of the deadly nature of oral

This article was published online on 5 May 2014. The affiliations were
incorrect. This notice is included in the online and print versions to indicate
that both have been corrected 22 May 2014.
*Corresponding author: C. Fuller, Department of OtolaryngologyHead and
Neck Surgery, Medical University of South Carolina, MSC 550, 135 Rutledge
Ave, Charleston, SC 29403. E-mail: cwfull@gmail.com
Additional Supporting Information may be found in the online version of this
article.

Results. Forty-eight articles satisfying inclusion criteria were identified.

Twenty-five were included in quantitative synthesis.
Conclusion. Oral cytology holds higher diagnostic value than specialists
oral examination, which holds higher value than in vivo toluidine blue staining. This study does not support the use of computer-aided or liquid-based
cytology. Future studies should be designed to test multiple methods in the
same patient population to allow direct comparison among various techniC 2014 Wiley Periodicals, Inc. Head Neck 00: 000000, 2014
ques. V

KEY WORDS: oral cancer, oral lesion, oral premalignancy, oral

screening, oral cancer, early detection

cancer and the accessibility of the oral cavity for testing,

early detection is an active field of research.
Diagnosis of oral cancer begins with a thorough history,
followed by conventional oral examination (COE). When
a suspicious lesion is identified, a full-thickness tissue
biopsy, such as punch biopsy, incisional biopsy, or excisional biopsy, is often performed as the gold standard
test for diagnosing oral cancer. However, these tests are
not without limitations. Biopsy usually requires a specialist and access to a pathologist familiar with oral cancer.
Many techniques have been advocated over the past several decades as aids or adjuncts in identifying which
lesions are suspicious enough to require biopsy. Unfortunately, there remains some confusion and controversy in
the literature about which, if any, of these adjunctive
techniques are most reliable.
In a 2008 review, Lingen et al5 made the important distinction between the screening and case-finding steps
in mucosal diagnostics. Screening is defined as an attempt
to detect unknown lesions, whereas case-finding is an
attempt to diagnose known lesions. This study is
restricted to determining the utility of case-finding techniques for oral cancer and dysplasia.
Case-finding techniques may have great benefit to primary care providers who must determine which patients
need referral to a specialist for definitive biopsy. This
systematic review and meta-analysis aims to elucidate the
utility and accuracy of adjunctive diagnostic techniques
for oral potentially malignant lesions.
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TABLE 1. List of inclusion criteria.

First phase inclusion criteria

la. Oral cancer adjunct tested against gold standard tissue biopsy
2a. 10 or more patients studied
3a. English language publications only
4a. Published in peer-reviewed journal
5a. Adult population only
Second phase inclusion criteria
lb. Gold standard used for definitive diagnosis in all patients or in
all patients within a prospectively-determined subgroup
2b. Adjunct attempts to diagnose dysplasia, rather than simply
indicating a correlation
3b. Quantitative data presented that allows for the abstraction of
232 contingency table (disease vs. test result)
4b. If multiple tests evaluated, clinical characteristics could not be
used to determine which adjunct was used
5b. Adjunct used prospectively on lesions without histologically
confirmed diagnosis
6b. Patients have no history of cancer
7b. No history or anticancer treatment for the lesion in question

METHODS
Literature search
Inclusion criteria for this systematic review were developed in two phases. Before conducting a literature search,
the first set of inclusion criteria were agreed upon
(Table 1).
The literature search strategy can be found in the Preferred Reporting Items for Systematic reviews and MetaAnalyses-compliant literature review diagram (Figure 1). A
PubMed search was performed by one author (C. F.) for
articles published between June 1993 and June 2013 using
the predetermined search terms (Appendix, online only;
Figure i). This search yielded a total of 2,488 references
when English-language and human subject constraints
were used. Eighty-eight additional articles were identified
by examining the reference lists of captured articles. A
Cochrane Library search (April 2008 to June 2013) was
also performed using the search term oral neoplasm,
which returned six references. References were screened
manually according to their titles and then based on
abstracts. 144 unique articles were identified that potentially satisfied the first five inclusion criteria.
After assessing the volume of articles pending further
review, additional inclusion criteria were established,
which are listed in Table 1. Additional articles obtained
from reference lists that were older than June 1993 would
be included in the systematic review, but not the metaanalysis.

Data extraction
Data from all articles included in the quantitative analysis were abstracted by 2 authors independently (C. F. and
R. C.). Data were only collected if a 2 3 2 contingency
table (test result vs presence of disease according to
biopsy) could be constructed from the data presented.
These data were double-checked against any stated summary statistics in each article, such as sensitivity, specific2

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ity, positive predictive value (PPV), negative predictive

value (NPV), and accuracy. In certain cases, the raw data
were reconstituted from these summary statistics by determining the whole number ratios that produced them. Data
reconstitution could be precisely achieved by solving five
equations for five unknown variables if sample size, sensitivity, specificity, PPV, and NPV were reported (Appendix, online only; Figure ii). When discrepancies arose, the
original articles authors were contacted for comment if
the article had been published in the last ten years. Failing this, consensus was reached among all authors regarding the validity of data inclusion.
Some inclusion criteria pertained to overall study
design, whereas others pertained to the characteristics of
the patients or their lesions (Table 1). When only some of
the patients satisfied these latter inclusion criteria, efforts
were made to extract only the data that described qualifying lesions or patients. When this was not possible, contact with the original authors was attempted if the article
was ten years old or less, and consensus was reached
among all authors.
When diagnostic test results were qualitative, we considered the presence of any level of concern for dysplasia
or cancer to be positive, including atypical,
equivocal, or any other result that implied the presence
of
malignancy/premalignancy;
results
such
as
inflammation or hyperplasia were considered negative, along with benign and normal results. Gold
standard results positive for cancer or any amount of dysplasia were also considered positive. In cases in which
articles used a different cutoff for defining positive and
negative test/biopsy results, contact with the original
authors was attempted if the article was ten years old or
less. If raw data could not be recovered, the authors definitions of positive and negative results were accepted.
This was only true of two of the included articles,6,7 both
of which reported the same study. This study defined the
cutoff between negative and positive to be between oral
intraepithelial neoplasia I and oral intraepithelial neoplasia II.
When test results were expressed quantitatively, the
authors cutoff for test results indicating dysplasia was
accepted as the cutoff for positivity, although the algorithm used to generate the cutoff was noted. When multiple algorithms were assessed, the dataset using the
algorithm with the highest accuracy was used to represent
the study in the analysis.
Where available, data on the results of COE were also
tabulated. Again, these data were only included when the
entire group of patients evaluated also underwent biopsy.
COE data was captured only when a benign versus not
benign lesion delineation was obvious. COE data was
excluded when the COE or visual impression declared an
area of mucosa to be normal, because this meta-analysis
evaluates the quality of adjuncts and COE in determining
the malignant potential of lesions, rather than normal
mucosa.
The cytology methods are prone to generating
inadequate or unreadable results. For these studies, an
additional analysis was performed to examine differences
in inadequacy rate between cytology methods. Only studies
that specifically reported the number of qualifying

DIAGNOSTIC

TECHNIQUES FOR ORAL LESIONS OF UNKNOWN MALIGNANT POTENTIAL

FIGURE 1. Preferred Reporting Items for

Systematic Reviews and Meta-Analyses
literature search flow diagram from original search through eventual inclusion of
qualifying data.

inadequate samples would be included for this part of the

analysis.

Statistical analysis
Once all contingency tables for each included study
were constructed, true and false-positive, true and falsenegative, and inadequate results were summed for each
group of datasets. For sensitivity calculations, the total
with dysplasia or cancer according to gold standard was
considered the sample size, whereas the proportion was
each studys sensitivity, to generate overall sensitivity and

95% confidence intervals (CIs). Similarly, for specificity

and accuracy, the sample size was the number of benign
lesions or total lesions (minus inadequate results), respectively, in each dataset, whereas the specificity or accuracy, respectively, was the proportion.
A meta-analysis of proportions was then conducted on
the sensitivity, specificity, and accuracy of each group and
subgroup, using MedCalc 12.7.0 (MedCalc Software bvba,
Belgium). Analysis of pooled proportions was performed
where appropriate. MedCalc used a FreemanTukey transformation8 (arcsine square root transformation) to calculate
the weighted summary proportion under the fixed and

FIGURE 2. Conventional oral

examination results. Forest plot
for sensitivity, specificity, and
accuracy of conventional oral
examination
datasets.
For
explanation of random versus
fixed effects models, see
Statistical Analysis subsection
in Methods section. [Color figure can be viewed in the online
issue, which is available at
wileyonlinelibrary.com.]

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FULLER ET AL.

TABLE 2. Diagnostic methods analyzed.

 Conventional Oral Examinationexamination conducted under normal, clinical fluorescent white lighting, without the use of stains, light filters or
magnification
 Oral Cytologythe microscopic examination of superficial oral tissue harvested by non-invasive means (including brush, spatula, curette, etc.)
 Toluidine Blue Stainingthe use of toluidine blue (i.e. tolonium chloride) dye to stain lesions in vivo, under conventional, white fluorescent lighting
 Laser-induced Autofluorescence Spectroscopythe use of specific excitation wavelengths to illicit detectable fluorescence spectra in native oral
lesions, without the use of photosensitizers such as 5-aminolevulinic acid, by any excitation wavelength or spectroscopic analysis algorithm
 Diffuse Reflectance Spectroscopythe use of photodetection to analyze the diffuse reflectance spectra of oral lesions, by any spectroscopic analysis algorithm

random effects models.9 Data were presented as weighted

proportions with corresponding 95% CIs. Both the fixed
effects model and the random effects model were used in
this study. Under the fixed effects model, it is assumed that
all studies come from a common population, and that the
effect size (the proportion) is not significantly different
among the different trials. This assumption is tested by the
heterogeneity test or I2 statistic. If this test yields a low
p value < .05, then the fixed effects model may be invalid.
In this case, the random effects model may be more appropriate, in which both the random variation within the studies and the variation between the different studies is
incorporated.
In addition, a chi-square test with Yates correction for
continuity was applied with two-sided (or two-tailed) p
values for the comparison of two proportions from independent samples expressed as a percentage, as calculated
from the aforementioned aggregations. For each aggregation, the random effects result was used when the I2 test
for heterogeneity yielded a p value < .05; otherwise, the
fixed effects result was used. Each group was weighted
according to the number of patients with dysplastic/malignant lesions, benign lesions, or total lesions with adequate
results, respectively. When the calculated p value was

<.05, the conclusion was that the two proportions were

significantly different.

RESULTS
A total of 48 articles were identified as eligible for
qualitative analysis,6,7,1056 37 of which were modern
era articles and, thus, eligible for inclusion in the aggregated quantitative analysis.6,7,1032,4556 One pair of
articles6,7 reported data from the same study, but, in this
case, the duplicate aided in the reconstitution of raw
data. The 37 modern articles reported 43 inclusion
criteria-satisfying datasets because many studies evaluated
multiple techniques, including all studies that reported
qualifying results of COE.
Data were included in the quantitative analysis and
aggregated for comparison for any techniques that had at
least three includable datasets. This left 25 articles across
five different adjuncts.6,7,1032 These techniques were COE
(three datasets), cytology (12 datasets), toluidine blue vital
staining (eight datasets), laser-induced autofluorescence
(LIAF) spectroscopy (four datasets) and diffuse reflectance
spectroscopy (DRS; three datasets). A total of 2,184 qualifying measurements were conducted in these studies on a

TABLE 3. Overall technique comparisons.

Sensitivity
Technique
DRS
LIAF
Cytology
COE
TB
Specificity
Technique
Cytology
LIAF
DRS
COE
TB
Accuracy
Technique
DRS
LIAF
Cytology
COE
TB

D1C
101
117
477
69
240

Sen
98.6
97.7
89.7
88.7
82.1

vs
vs
vs
vs
vs

TB
<0.001
<0.001
0.006
0.263
-

COE
0.014
0.026
0.965
-

Cytology
0.007
0.010
-

LIAF
0.990
-

Ben
670
23
21
80
349

Spe
89.8
84.4
83.2
60.9
52.8

vs
vs
vs
vs
vs

TB
<0.001
0.006
0.013
0.235
-

COE
<0.001
0.064
0.098
-

DRS
0.540
0.763
-

LIAF
0.626
-

N
122
140
1147
149
589

Acc
96.5
95.9
85.7
77.6
66.7

vs
vs
vs
vs
-

TB
<0.001
<0.001
<0.001
0.014
-

COE
<0.001
<0.001
0.014
-

Cytology
0.001
0.001
-

LIAF
0.944
-

Abbreviations: D1C, number of dysplasias and cancers by gold standard; Sen, sensitivity; COE, conventional oral exam; TB, toluidine blue; LIAF, laser-induced autofluorescence; DRS, diffuse reflectance spectroscopy; Ben, number of benign lesions by gold standard; N, overall sample size (minus inadequate test results); Acc, accuracy).
Overall comparisons proportion using calculated values of sensitivity, specificity and accuracy from fixed or random effects models (see Appendix) and number of dysplastic/cancerous, benign and
total lesions, respectively. Techniques are ordered in descending quality. Statistically significant values are bolded and italicized, favoring the technique in the left-hand column.

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TECHNIQUES FOR ORAL LESIONS OF UNKNOWN MALIGNANT POTENTIAL

TABLE 4. Subgroup comparisons.

Sen, D1C
Spe, Ben
Acc, N
Sen, D1C
Spe, Ben
Acc, N
Sen, D1C
Spe, Ben
Acc, N
Sen, D1C
Spe, Ben
Acc, N
Sen, D1C
Spe, Ben
Acc, N
Sen, D1C
Spe, Ben
Acc, N

Technique

p-value

Technique

Wooden spatula cytology (A)

77.8%, 9
100%, 71
97.5%, 80
OralCDx (C)
94.3%, 160
83.4, 286
84.9%, 446
Sciubba (OralCDx) (E)
100%, 102
92.9%, 196
95.3%, 298
Toluidine blue application (G)
84.8%, 171
53.9%, 53.9
69.0%, 339
Toluidine blue Mashberg (I)
82.9%, 26
54.5%, 67
63.2%, 93
LIAF (dorsal/ventral tongue) (K)
98.3%, 26
50.0%, 2
94.3%, 28

vs.
0.489
0.004
0.003
vs.
0.067
0.026
0.859
vs.
0.001
<0.001
<0.001
vs.
0.109
0.611
0.226
vs.
0.935
0.816
0.444
vs.
0.643
0.706
0.870

Brush cytology (B)

90.4%, 468
88.0%, 599
84.4%, 1067
Conventional brush cytology (D)
88.6%, 308
89.9%, 313
84.1%, 621
Recent OralCDx (F)
87.8%, 58
76.8%, 90
77.6%, 148
Toluidine blue rinse (H)
75%, 69
50.6%, 181
63.9%, 250
Toluidine blue non-Mashberg (J)
81.4%, 214
52.0%, 282
67.9%, 496
LIAF (other locations) (L)
97.2%, 57
87.7%, 20
96.1%, 64

Abbreviations: Sen, sensitivity; D1C, number of dysplasias and cancers by gold standard; Spe, specificity; Ben, number of benign lesions by gold standard; Acc, Accuracy; N, sample size; LIAF,
laser-induced autofluorescence).
Direct comparison of proportions between dataset subgroups, using the overall sensitivity, specificity and accuracy calculated from fixed or random effects models (see figures). P-values indicating
significant differences are listed in bold print, with the better technique also listed in bold print. (Studies composing these subgroups are listed in Appendix Tables i-v).

total of 1,887 unique qualifying lesions. Appendix Tables i

to v, online only, summarize these studies. A brief explanation of each of the techniques evaluated by articles included
in our quantitative synthesis can be found in Table 2. Further basic science discussions of these techniques are
included in Appendix section A, online only.
The remaining 24 articles analyzed the value of many
additional techniques, or were published before 1993 and,
thus, not considered modern era. However, the findings
from these articles, including sensitivity, specificity, and
accuracy are summarized in Appendix Table vi, online only.
Direct comparisons between each of the 5 techniques
are found in Table 3. A set of sample Forest plots for
COE is found in Figure 2. The remaining Forest plots are
presented in Appendix Figures iii to vi, online only. A
more detailed discussion of the results of the metaanalysis can be found in Appendix section C, online only.
The most sensitive technique was DRS (98.6%), followed closely by LIAF (97.7%; Table 3). These techniques each demonstrated significantly better sensitivity
than the remaining three techniques (COE, cytology, and
toluidine blue staining). The least sensitive technique was
toluidine blue (82.1%), which was significantly worse
than DRS, LIAF, and cytology.
The most specific technique was cytology (89.8%),
whereas the least specific technique was toluidine blue
(52.8%). Toluidine blue was less specific than any other
technique except COE.
The most accurate tests were DRS and LIAF (96.5%
and 95.9%, respectively). They were each more accurate
than any of the remaining three techniques. Toluidine
blue was the least accurate test (66.7%) and was signifi-

cantly outperformed in this category by the four other

tests. Within each technique category, subgroups were
established to investigate comparisons between variations
in technique (Table 4).
The single cytology dataset presenting results for
wooden spatula harvesting demonstrated significantly
higher specificity and accuracy compared to brush biopsy.
Conventionally read brush biopsy outperformed
computer-assisted brush biopsy based on specificity only.
The older, corporate-sponsored OralCDx trial demonstrated significantly higher sensitivity, specificity, and
accuracy than the two newer OralCDx datasets collectively. No other subgroup comparisons demonstrated statistically significant differences.

DISCUSSION
In discussing the implications of this analysis, the authors
first wish to address its limitations. The most important
limitation of this meta-analysis is the variability in the
patient population between studies. This is not only important with regard to patient demographics, but also the
lesions they present. Lesions that seem to be frankly cancerous should be referred immediately to a surgeon and
biopsied or excised, whereas lesions that have obviously
nonmalignant etiologies instead warrant observation and
close follow-up; diagnostic adjuncts are most valuable in
assessing the lesions that fall in the wide gap between these
two categories, and the less experienced the examiner, generally the wider the gap. Although the inclusion criteria
were intended to limit data only to lesions that had not
been definitively diagnosed by biopsy, this still leaves a
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FULLER ET AL.

vast range of clinical possibilities, for some of whom a

diagnostic adjunct may be inappropriate.
Because it is very likely that the research studies were
conducted more often by specialists with an interest in
oral cavity cancer than by general clinicians, the examiners that determined subject inclusion were also likely to
have excellent clinical judgment in determining potentially dysplastic lesions. This may limit the applicability
of this data to general medical settings.
Early detection of oral cancer is rightfully an important
focus of research. The impact of earlier diagnosis for all
cancers is obvious, but is of particular value with regard to
the oral cavity because of reduced survival and quality of
life that occurs with later diagnosis. This persists despite
the accessibility of the oral cavity to direct examination.
Yet there still remains considerable debate as to the clinical
value of various oral cancer diagnostic adjuncts for clinicians at various levels of specialization. There are two
main clinical questions at issue; (1) What resources should
a general dentist or general medical practitioner bring to
bear when confronted by a lesion of uncertain malignant
potential; and (2) What resources are valuable for a cancer
specialist who may have the same question?
The former question is critical from a broad epidemiologic standpoint, especially in the third world or even
among rural general clinicians in developed nations. The
ideal test for the general practitioner would be one that is
inexpensive, noninvasive, sensitive, and expedient. However, even the judgment of specialists may be aided by
the use of these adjunctive tools, because the higher volume of potentially malignant lesions in their practice
would allow an adjunct to potentially save a larger number of patients from unnecessary biopsy.
The merits and drawbacks of each of the included techniques are discussed below, considering the data presented in the meta-analysis.

Conventional oral examination

To the authors knowledge, this is the first metaanalysis evaluating prospective clinical oral examinations
made by experts in oral cancer against the gold standard
of tissue biopsy.
One limitation of the COE data specifically is the
observer effect (or so-called Hawthorn effect) upon the
clinical examination. However, in addition to the behavioral changes that occurred simply because of the knowledge that one is being observed, there may have been some
semiconscious change to the evaluation. Because the evaluator knew that all of the lesions would receive a biopsy
anyway, the risk of a missed cancer diagnosis because of
an examiners error was completely ameliorated by each
patients inclusion in the prospective studies. There is a
chance that this changed the subjective cutoff for each
assessment to one that may not be used by that evaluator
clinically.

Cytology and noninvasive tissue sampling

Based on the meta-analytic data alone, it seems that
noninvasive cytology is quite useful. We demonstrate a
statistically significantly improved specificity and accuracy of cytology over a COE by a cancer specialist.
6

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Despite this diagnostic improvement, in rural or underserved areas, the use of this technique could delay more
definitive diagnosis by a week or more while the specimen is in transit and in process. Furthermore, of the two
main clinically available adjuncts analyzed here (the other
being vital staining), cytology is considerably more
expensive. Out-of-pocket cost charged by CDx Diagnostics for OralCDx sample analysis is $225. According to
the authors institution chargemaster, the out-of-pocket
cost for conventionally read oral smear cytology is $186,
whereas the out-of-pocket cost for conventionally read
liquid-based cytology is $300. Meanwhile, a 100-mL bottle of 1% toluidine blue can be purchased at retail for
about $15.
This study demonstrated a significantly better specificity by conventional cytology compared to the proprietary
computer-based OralCDx system. The data also demonstrate significant differences in the sensitivity, specificity,
and accuracy of the initial corporate-sponsored trial14 and
the pooled sensitivity of older trials (Table 4).20,21
Clinicians must interpret the preceding data with caution. An important limitation of the comparison between
OralCDx and conventional cytology is the nature of the
inclusion criteria. Disease progression affects sensitivity
and specificity (typically later-stage disease is easier to
diagnose) even if prevalence does not. The two more
recent Oral CDx articles specifically excluded any
patients who were diagnosed on examination with frankly
cancerous lesions, and one of them also excluded clinically diagnosed dysplasias. This likely contributed to the
statistically significant difference in sensitivity between
this dataset and the dataset published by Sciubba et al,14
in which biopsies were performed only on patients prospectively determined likely to have cancer. This underlines the limitation stated above regarding population
heterogeneity. In addition, spatula sampling of oral
lesions was demonstrated valuable by this meta-analysis.
Only one article evaluated non-Papanicolaou/hematoxylin-eosin staining methods,22 and only one article evaluated
liquid-based cytology.19 The small sets of data presented
by these studies (N 5 20 and 26, respectively) were not of
sufficient statistical power to demonstrate statistical significance, even if such a difference exists (data not shown).
We consider acridine orange staining and liquid-based
cytology to offer promise as a future research topic, but this
meta-analysis does not support their routine use. This metaanalysis could not demonstrate any statistically significant
difference in inadequacy rate among cytology methods.

Vital staining techniques

Despite its affordability, according to this analysis and
review, toluidine blue staining does not improve on an
oral examination by a cancer specialist once a lesion has
been identified. In fact, its use seems to obscure the
examination results because the COE accuracy was significantly better than the accuracy of toluidine blue. Toluidine blue was also outperformed based on accuracy by
every other technique in the quantitative synthesis.
However, this does not mean that the stain has no value
at all. The value of the dye as an oral cancer screening
tool was not evaluated in this study. There were also no
data on the accuracy of a clinical examination performed

DIAGNOSTIC

by a general medical or dental practitioner, and there may

be a use for the dye in the hands of a generalist. However, the staining pattern in each of the studies was also
likely interpreted by an expert in oral cancer rather than a
generalist who may also be a poorer judge of staining
results.

TECHNIQUES FOR ORAL LESIONS OF UNKNOWN MALIGNANT POTENTIAL

ning to screen for unknown lesions, this drawback may not

significantly impact its clinical value. This analysis shows
no significant difference between the diagnostic accuracy
of LIAF for lesions in the dorsal and lateral tongue and its
accuracy for lesions elsewhere.

Avenues for future research

Laser-induced autofluorescence and diffuse reflectance
spectroscopy
This meta-analysis identified a total of five studies
using the LIAF and/or DRS. Four of these studies shared
one author, and three of them had the same primary
author. The homogeneity of authorship for these techniques severely limits the generalizability of the metaanalytic data. The primary author for three of these studies29,30,32 confirmed the lack of patient overlap between
studies evaluating the same adjunct.
Perhaps the biggest caveat for interpreting the spectroscopic data is the retrospective nature of model construction inherent to the development of any new diagnostic
test. Only a single study29 validated their constructed
model prospectively on a second set of undiagnosed
lesions. We strongly encourage future researchers to adopt
this method of model validation, as it is the best way to
demonstrate the applicability of the adjunct to actual
patients.
Nonetheless, the data from these techniques were impressive. By sensitivity and overall accuracy, they outperformed all other techniques here presented (Table 3).
Accuracy data should also be interpreted with caution. The
apparent accuracy of a test (defined as true results/total
results) can be affected by the prevalence of disease, especially when a tests sensitivity and specificity are appreciably different from one another. For example, a highly
sensitive but poorly specific test would look more accurate
when used on a patient population with high prevalence of
disease. This would have a greater effect on the accuracy
of the spectroscopic studies, because their prevalence of
disease was very high, and these techniques seem to be
more sensitive than they are specific.
Before declaring that these techniques are the wave of
the future for oral cancer diagnosis, further study verifying these models prospectively is needed. However, the
success of the spectroscopic diagnostic strategies is
encouraging compared to the remaining three methods.
Spectroscopy has the potential to offer a noninvasive
instantaneous answer to the clinical question of whether a
lesion is benign, dysplastic, or cancerous. The question of
whether these can be successfully brought to bear in a
clinical setting may rest on the systems financial accessibility to the clinicians that would benefit most from using
them: rural and general clinician.
It is commonly noted that LIAF has limited use in lesions
of the lateral and dorsal tongue and vermillion border of
the lip because normal mucosa in these areas generates a
fluorescence spectrum analogous to that of cancer.
Although this was demonstrated in one of the included
articles,30 the diagnostic errors were almost completely
confined to normal, lesion-free mucosa. We note that
because these spectroscopic techniques are under development for use in evaluating known lesions, rather than scan-

Considering the lower incidence rate of oral cancer

compared to other forms of malignancy, further study in
the area of known lesion diagnostics may hold the key to
reducing the mortality of this disease. Future research
should facilitate the evaluation of diagnostic adjuncts
against one another in the same patient population, with
confirmation in all patients by tissue biopsy. In addition,
the reporting of CIs or SEs alongside descriptive statistics, such as sensitivity, specificity, PPV, NPV, accuracy,
and inadequacy rate is strongly encouraged. Without these
measures of confidence, comparisons between diagnostic
modalities hold far less value.
Future meta-analyses on this topic may continue to prove
quite valuable, as the sample sizes required to achieve statistical significance may outstrip the resources of any one
researcher or research group. Publication of raw data or its
inclusion in appendices for meta-analytic purposes is
important.
Researchers need to consider the examination skills of
the clinicians that are most likely to benefit from these
adjuncts. To evaluate whether any of these adjuncts are
useful to general dentists and medical doctors, the baseline
general practitioner COE must be studied against gold
standard biopsy. Future research methods should consider
defining their examination-based inclusion criteria by the
generalists examination, if these clinicians are the ones
who will most benefit from the use of the adjunct.

CONCLUSION
This meta-analysis demonstrates a statistically significant
improvement in diagnostic quality of oral cytology methods over COE based on superior specificity and overall
accuracy. This analysis does not support the use of more
costly versions of oral cytology, such as OralCDx
computer-assisted cytology or liquid-based cytology based
on diagnostic accuracy or inadequacy of the sample. Other
sampling techniques for cytology may hold promise, but
further study is needed to assess their diagnostic value. For
visible lesions, toluidine blue seems to hold little value.
Few head-to-head trials have been published evaluating different techniques in the same population.
Both LIAF and DRS had a significantly higher accuracy
than COE and toluidine blue staining, and significantly better sensitivity than toluidine blue staining. DRS also demonstrated significant improvement in sensitivity compared
to cytology. These diagnostic adjuncts are promising and
we encourage further research prospectively evaluating
these methods using established diagnostic algorithms.

Acknowledgments
The authors thank Drs. HohlwegMajert,18 Kaczmarzyk
and Mojsa,31 Narayanan and Mallia,29,30,32 and Joseph
Califano and David Sirois for their assistance with interpreting and clarifying data.
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REFERENCES
1. Petersen PE. Strengthening the prevention of oral cancer: the WHO perspective. Community Dent Oral Epidemiol 2005;33:397399.
2. American Cancer Society. Cancer Facts and Figures 2013. Available at: http://
www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/
document/acspc-036845.pdf. Accessed August 1, 2013.
3. Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.
cancer.gov) SEER*Stat Database: Mortality All COD, Aggregated With
State, Total U.S. (19692010) <Katrina/Rita Population Adjustment>,
National Cancer Institute, DCCPS, Surveillance Research Program, Surveillance Systems Branch, released April 2013. Underlying mortality data
provided by NCHS (www.cdc.gov/nchs).
4. Surveillance, Epidemiology, and End Results (SEER) Program (www.seer.
cancer.gov) Research Data (19732010), National Cancer Institute,
DCCPS, Surveillance Research Program, Surveillance Systems Branch,
released April 2013, based on the November 2012 submission.
5. Lingen MW, Kalmar JR, Karrison T, Speight PM. Critical evaluation of
diagnostic aids for the detection of oral cancer. Oral Oncol 2008;44:1022.
6. Navone R, Burlo P, Pich A, et al. The impact of liquid-based oral cytology
on the diagnosis of oral squamous dysplasia and carcinoma. Cytopathology
2007;18:356360.
7. Navone R. Cytology of the oral cavity: a re-evaluation. Pathologica 2009;
101:68.
8. Freeman MF, Tukey JW. Transformations related to the angular and the
square root. Ann Math Stat 1950;21:607611.
9. DerSimonian R, Laird N. Meta-analysis in clinical trials. Control Clin Trials 1986;7:177188.
10. Guneri P, Epstein JB, Kaya A, Veral A, Kazandi A, Boyacioglu H. The
utility of toluidine blue staining and brush cytology as adjuncts in clinical
examination of suspicious oral mucosal lesions. Int J Oral Maxillofac Surg
2011;40:155161.
11. Gillenwater A, Jacob R, Ganeshappa R, et al. Noninvasive diagnosis of
oral neoplasia based on fluorescence spectroscopy and native tissue autofluorescence. Arch Otolaryngol Head Neck Surg 1998;124:12511258.
12. Koch FP, Kaemmerer PW, Biesterfeld S, Kunkel M, Wagner W. Effectiveness of autofluorescence to identify suspicious oral lesionsa prospective,
blinded clinical trial. Clin Oral Investig 2011;15:975982.
13. Erenmemisoglu A, Ustun H, Kartal M. Carcinoma of buccal mucosa in
smokeless tobacco users: a preliminary study of the use of cytology for
early detection. Cytopathology 1995;6:403408.
14. Sciubba JJ. Improving detection of precancerous and cancerous oral
lesions. Computer-assisted analysis of the oral brush biopsy. U.S. Collaborative OralCDx Study Group. J Am Dent Assoc 1999;130:14451457.
15. Maraki D, Becker J, Boecking A. Cytologic and DNA-cytometric very
early diagnosis of oral cancer. J Oral Pathol Med 2004;33:398404.
16. Gupta A, Singh M, Ibrahim R, Mehrotra R. Utility of toluidine blue staining and brush biopsy in precancerous and cancerous oral lesions. Acta
Cytol 2007;51:788794.
17. Mehrotra R, Singh MK, Pandya S, Singh M. The use of an oral brush
biopsy without computer-assisted analysis in the evaluation of oral lesions:
a study of 94 patients. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2008;106:246253.
18. HohlwegMajert B, Depp H, Metzger MC, et al. Sensitivity and specificity
of oral brush biopsy. Cancer Invest 2009;27:293297.
19. Delavarian Z, Mohtasham N, MosannenMozafari P, Pakfetrat A, Shakeri
MT, GhafoorianMaddah R. Evaluation of the diagnostic value of a modified liquid-based cytology using OralCDx brush in early detection of oral
potentially malignant lesions and oral cancer. Med Oral Patol Oral Cir
Bucal 2010;15:e671e676.
20. Koch FP, Kunkel M, Biesterfeld S, Wagner W. Diagnostic efficiency of
differentiating small cancerous and precancerous lesions using mucosal
brush smears of the oral cavitya prospective and blinded study. Clin
Oral Investig 2011;15:763769.
21. Mehrotra R, Mishra S, Singh M, Singh M. The efficacy of oral brush
biopsy with computer-assisted analysis in identifying precancerous and
cancerous lesions. Head Neck Oncol 2011;3:39.
22. Prakash N, Sharada P, Pradeep GL, Soundarya N. Reliability of acridine
orange fluorescence microscopy in oral cytodiagnosis. Indian J Dent Res
2011;22:649653.
23. Onofre MA, Sposto MR, Navarro CM. Reliability of toluidine blue application in the detection of oral epithelial dysplasia and in situ and invasive
squamous cell carcinomas. Oral Surg Oral Med Oral Pathol Oral Radiol
Endod 2001;91:535540.
24. Nagaraju K, Prasad S, Ashok L. Diagnostic efficiency of toluidine blue
with Lugols iodine in oral premalignant and malignant lesions. Indian J
Dent Res 2010;21:218223.
25. Upadhyay J, Rao NN, Upadhyay RB, Agarwal P. Reliability of toluidine
blue vital staining in detection of potentially malignant oral lesionstime
to reconsider. Asian Pac J Cancer Prev 2011;12:17571760.
26. CancelaRodrguez P, CereroLapiedra R, EsparzaG
omez G, Llamas
Martnez S, Warnakulasuriya S. The use of toluidine blue in the detection of premalignant and malignant oral lesions. J Oral Pathol Med 2011;40:300304.
27. Awan KH, Yang Y, Morgan P, Warnakulasuriya S. Utility of toluidine blue
as a diagnostic adjunct in the detection of potentially malignant disorders

HEAD & NECKDOI 10.1002/HED

MONTH 2014

28.

29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.

46.
47.
48.
49.
50.
51.

52.
53.
54.
55.
56.

of the oral cavitya clinical and histological assessment. Oral Dis 2012;
18:728733.
Mojsa I, Kaczmarzyk T, Zaleska M, Stypulkowska J, ZapalaPospiech A,
Sadecki D. Value of the ViziLite Plus System as a diagnostic aid in the
early detection of oral cancer/premalignant epithelial lesions. J Craniofac
Surg 2012;23:e162e164.
Mallia RJ, Thomas SS, Mathews A, et al. Laser-induced autofluorescence
spectral ratio reference standard for early discrimination of oral cancer.
Cancer 2008;112:15031512.
Mallia RJ, Narayanan S, Madhavan J, et al. Diffuse reflection spectroscopy: an alternative to autofluorescence spectroscopy in tongue cancer
detection. Appl Spectrosc 2010;64:409418.
Jayanthi JL, Subhash N, Stephen M, Philip EK, Beena VT. Comparative evaluation of the diagnostic performance of autofluorescence and diffuse reflectance
in oral cancer detection: a clinical study. J Biophotonics 2011;4:696706.
Mallia R, Thomas SS, Mathews A, et al. Oxygenated hemoglobin diffuse
reflectance ratio for in vivo detection of oral pre-cancer. J Biomed Opt
2008;13:041306.
Silverman S Jr, Becks H, Farber SM. The diagnostic value of intraoral
cytology. J Dent Res 1958;37:195205.
Tiecke RW, Kendrick FJ, Calandra JC. Smear techniques in the diagnosis
of intra-oral carcinoma. Dental Prog (Chic) 1961;1:192198.
Hellinger MJ, Karpas CM, Sellers W Jr. A clinicopathologic correlation of
oral white lesions. Study of forty-five cases. Oral Surg Oral Med Oral
Pathol 1963;16:13561376.
Gardner AF. An investigation of the use of exfoliative cytology in the diagnosis of malignant lesions of the oral cavity. The cytologic diagnosis of
oral carcinoma. Acta Cytol 1964;8:436445.
Caulder SL. Fluorescence microscopy utilizing acridine orange in oral
cytodiagnosis. Oral Surg Oral Med Oral Pathol 1967;23:343350.
Gaither WD. Comparison of exfoliative cytodiagnosis and histodiagnosis
of oral lesions: review of the literature and report of 75 cases. J Oral Surg
1967;25:446454.
Reddy CR, Ramulu C, Sundareshwar B, Raju MV, Gopal R, Sarma R.
Toluidine blue staining of oral cancer and precancerous lesions. Indian J
Med Res 1973;61:11611164.
Mashberg A. Reevaluation of toluidine blue application as a diagnostic
adjunct in the detection of asymptomatic oral squamous carcinoma: a continuing prospective study of oral cancer Ill. Cancer 1980;46:758763.
Mashberg A. Final evaluation of tolonium chloride rinse for screening of
high-risk patients with asymptomatic squamous carcinoma. J Am Dent
Assoc 1983;106:319323.
Mashberg A. Tolonium (toluidine blue) rinsea screening method for recognition of squamous carcinoma. Continuing study of oral cancer IV.
JAMA 1981;245:24082410.
Silverman S Jr, Migliorati C, Barbosa J. Toluidine blue staining in the
detection of oral precancerous and malignant lesions. Oral Surg Oral Med
Oral Pathol 1984;57:379382.
Silverman S Jr, Migliorati C. Toluidine blue staining and early detection of
oral precancerous and malignant lesions. Iowa Dent J 1992;78:1516.
Chen YW, Lin JS, Wu CH, Lui MT, Kao SY, Fong Y. Application of in
vivo stain of methylene blue as a diagnostic aid in the early detection and
screening of oral squamous cell carcinoma and precancer lesions. J Chin
Med Assoc 2007;70:497503.
Kulapaditharom B, Boonkitticharoen V. Performance characteristics of fluorescence endoscope in detection of head and neck cancers. Ann Otol Rhinol Laryngol 2001;110:4552.
Seetharam SS, Ramachandran CR. Fine needle aspiration cytology as a
diagnostic test for oral squamous cell carcinoma. Oral Dis 1998;4:180186.
Sharwani A, Jerjes W, Salih V, et al. Fluorescence spectroscopy combined
with 5-aminolevulinic acid-induced protoporphyrin IX fluorescence in
detecting oral premalignancy. J Photochem Photobiol B 2006;83:2733.
Sharwani A, Jerjes W, Salih V, et al. Assessment of oral premalignancy
using elastic scattering spectroscopy. Oral Oncol 2006;42:343349.
Farah CS, McCullough MJ. A pilot case control study on the efficacy of
acetic acid wash and chemiluminescent illumination (ViziLite) in the visualisation of oral mucosal white lesions. Oral Oncol 2007;43:820824.
Nieman LT, Kan CW, Gillenwater A, Markey MK, Sokolov K. Probing
local tissue changes in the oral cavity for early detection of cancer using
oblique polarized reflectance spectroscopy: a pilot clinical trial. J Biomed
Opt 2008;13:024011.
Tsai MT, Lee HC, Lee CK, et al. Effective indicators for diagnosis of oral
cancer using optical coherence tomography. Opt Express 2008;16:
1584715862.
Bremmer JF, Graveland AP, Brink A, et al. Screening for oral precancer with
noninvasive genetic cytology. Cancer Prev Res (Phila) 2009;2:128133.
Pentenero M, Gieretti W, Navone R, et al. DNA aneuploidy and dysplasia
in oral potentially malignant disorders: association with cigarette smoking
and site. Oral Oncol 2009;45:887890.
Amelink A, Sterenborg HJ, Roodenburg JL, Witjes MJ. Non-invasive measurement of the microvascular properties of non-dysplastic and dysplastic oral
leukoplakias by use of optical spectroscopy. Oral Oncol 2011;47:11651170.
Saeki N, Tsuzuki K, Negoro A, et al. Utility of real-time diagnosis using
contact endoscopy for oral and lingual diseases. Auris Nasus Larynx 2011;
38:233239.