The Value of Toluidine Blue Staining Test in Assessing Disease

Free Margins of Oral Cavity Carcinomas

DRAGOS CRISTIAN STEFANESCU1, OCTAVIAN CEACHIR2,3, VIOREL ZAINEA2,3, MURA HAINAROSIE2, CATALINA PIETROSANU2*,
IRINA GABRIELA IONITA2, RAZVAN HAINAROSIE2,3
1
Gen. Dr. Aviator Victor Anastasiu Institute of Aeronautical and Spatial Medicine, 88th Mircea Vulcanescu Str., 010825, Bucharest,
Romania
2
Carol Davila University of Medicine and Pharmacy, 8th Eroii Sanitari Blvd., 050474, Bucharest, Romania
3
Prof. Dr. D. Hociota Institute of Phonoaudiology and Functional ENT Surgery, 21 st Mihail Cioranu Str., 050751, Bucharest,
Romania

Oral cancer is one of the most frequent head and neck cancers. It is responsible for about 48% of the
carcinomas located in this region. The management of the oral cancer is difficult to perform because the
diagnosis usually occurs in a late stage of the disease. Surgical excision of the tumor with free margins is
mandatory in order to obtain a good survival rate, but due to the locally advanced disease, it may be difficult
to achieve. The aim of the paper is to determine the utility of the toluidine blue staining test in assessing
disease free resection margins during the transoral approach of oral carcinomas.
Key words: oral carcinoma, toluidine blue solution, disease free margins

Head and neck carcinomas represent almost 3% of all Experimental part

cancer types [1,2]. The incidence of oral, lip and pharyngeal
cancer in Romania is 15.5 (100,000), ranking third place in
Europe after Hungary and Slovakia. The mortality is 9.1
(100,000), again reaching the third place in Europe. The
oral cancers represent 48% of all head and neck cancers,
squamous cell carcinoma accounting for nearly 90% of
the cases, more than 300,000 new cases being discovered
around the world.
Oral cancer has a global incidence of 10 million cases.
The expected number for the year 2020 will be around 15
millions cases. The oral carcinoma survival rate is around
50% and it has not been improved in the last 50 years
because of the late stage diagnosis [3].
The management of the oral carcinomas is primarily
accomplished by surgical excision of the tumor and
thorough neck nodes dissection followed by adjuvant radio
chemo therapy.
The surgical approach of the oral carcinoma tends to be
trans orally due to the new ablative technologies available
on the market as CO2 LASER fiber, radiofrequency and
coblation [4].
The disease free resection margins are sometimes
difficult to asses [5,6], but mandatory when using transoral
surgery. So far, frozen sections have been the standard in
determining disease free resection margins, due to the high
sensitivity (88%) and specificity (99.6%) of the method.
Sometimes the frozen sections method may not be
available, and we have to consider the time that is needed
for this type of diagnostic. All in all, this method will add to
the surger y duration and will raise the cost of the
intervention for the hospital.
The aim of the paper is to evaluate the value of the
toluidine blue staining test in determining the disease free
margins in oral carcinomas approached trans orally [7]. It
is a simple and reliable test that had gained
acknowledgement in the past years due to the fast and
low cost reliable results [8,9].
The study was performed in 2 centers – The Institute of
Phonoaudiology and Functional ENT Surgery Prof Dr D
Hociota and Gen. Dr. Aviator Victor Anastasiu Institute of
Aeronautical and Spatial Medicine from Bucharest.
We selected a group of 32 T1 to T4 patients with
confirmed scuamous cell carcinoma located in the oral
region. The tumor was located at the level of the tongue
(25%), palatine tonsils (31.25%), retromolar trigone
(15.62%), velar area (9.37%) and floor of the mouth
(18.75%) (table 1). In the study we included 25 men and 7
women with ages between 57 to 72 year old. We excluded
the patients that have a history of oral carcinoma or for
which radiotherapy was previously performed, due to the
fact that extensive inflation or scar tissue will lead to
difficult interpretation of the staining image.
We performed transoral surgery for all the patients and
neck nodes excision according to the staging of the tumor.
We focused on the disease free resection margins.
The staining protocol was preformed as following: the
tumor was irrigated with saline solution, after aspiration
the tumoral area was washed with acetic acid 1%. The
tumoral area was then dried with a gauze.
Toluidine blue solution 1% was applied with a cotton on
the tumor and on the surrounding area and left in place for
30 s. After that the area was washed with 1% acetic acid.
The area was then dried with a gauze.
The tumoral extension and tumoral halo were carefully
examined. The surfaces coloured in dark blue were
considered to be positive and in that manner we could
determine the needed resection area.
Transoral surgery was performed using Lumenis CO2
LASER in 18 cases, Elman 4 Mhz Radiofrequency in 12
cases and Arthrocare coblation wand in 2 cases. According
to the cancer staging, in the same surgery, we performed
bilateral neck nodes dissection.
After tumor removal and hemostatic maneuvers we tried
to determine if the resection margins were correctly

* email: catapietrosanu@gmail.com; Tel.: 0723627405

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respected. We applied the above staining protocol with
toluidine blue. If dark blue margins were determined in the
resection of the respective area we considered that the
margins are not disease free and we must continue with
the resection until on toluidine blue staining we don’t have
dark blue colored margins anymore. In such case we could
consider that the resection of the tumor was performed
with disease free margins.
From the resection margins we harvested 10 areas
oriented according to the tumor morphology and send them
to histopathology department. The last resected areas were
also sent to histopathology department.
We then compared the results of the histopahological
findings with the results obtained by toluidine blue staining
test.
Results and discussions
Not one probe of the disease free margins obtained using
the toluidine blue method was disease positive, so we have
concluded that the toluidine blue staining test had 100%
sensitivity. From the positive resection margins on toluidine
blue only 83% confirmed as real positive at the histo-
pathology department.
Conclusions
Surgical resection with disease free margins remains
the main option of treatment in oral carcinomas. The
complete resection of the tumor and complete resection
of the neck nodes influences dramatically the loco-regional
recurrence of the tumor and the survival rate.
Frozen sections are sometimes not available in all the
surgical centers and even if they are available, it is a time
consuming technique that will add important costs for the
hospital. Further more it is proved that frozen sections will
not provide the surgeon with 100% disease free margins
because the technique is not 100% accurate and is not
able to completely eradicate positive margins
The toluidine blue staining test is a reliable, minimally
invasive, simple and inexpensive test that is easy to use. It
1256 http://www.revistadechimie.ro
Table 1
ORAL CARCINOMA LOCATION IN
THE STUDY GROUP
is not a time consuming technique, but it is able to provide
high sensitivity results.
The toluidine blue staining test did not provide false
negative disease free margins, thus enabling the surgeon
to perform a correct resection of the tumor. We have had
false positive margins in our biopsies but we think that the
reason was the manipulation of the tissue during surgery
or the use of ablative technologies such as LASER,
radiofrequency or coblation, all of this technologies having
probably produced histological modifications due to the
thermal effect on the tissue.
Toluidine blue staining test is a reliable, inexpensive test
that can be used routinely. It is still mandatory to analyze in
the histopathological department the disease free margins
in order to determine that the tumoral resection was correct
or not.
Further studies must be made in order to assess if the
resection margins stained with toluidine blue are observed
in narrow band light (NBI) so we can improve our results.
Acknowledgement: All authors have contributed equally to this
manuscript.
References
1.MIGNOGNA MD, FEDELE S, LO RUSSO L. Eur J Cancer Prev 2004; 13,
p. 139-42.
2.ORD RA, AISNER S. J Oral Maxillofac Surg 1997; 55, p. 663-71.
3.MASHBERS A, SAMIT A. CA Cancer J Clin 1995; 45, p. 328-51.
4.SHAH JP, GIL Z. Oral Oncol 2009; 45, p. 394-401.
5.LOREE TR, STRONG EW. Am J Surg 1990; 160, p. 410-4.
6.DINARDO LJ, LIN J, KARAGEORGE LS, POWERS CN. Laryngoscope
2000; 110, p. 1773-6.
7.SIDDIQUI IA, FAROOQ MU, SIDDIQUI RA, RAFI SMT. Pak J Med Sci.
2006; 22, p. 184-7.
8.EPSTEIN JB, SCIUBBA J, SILVERMAN S JR, SROUSSI HY. Head Neck
2007; 29, p. 948-58.
9.PORTUGAL LG, WILSON KM, BIDDINGERPW, GLUCKMANl JL. Arch
Otolaryngol Head Neck Surg 1996; 122, p. 517-9.
Manuscript received: 23.03.2016
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