Colorimetric analysis of unstained lesions surrounding oral squamous cell carcinomas and oral potentially malignant disorders using iodine

Abstract

To determine whether the measurement of staining with 3% Lugol’s solution provided efficient criteria for determining the area of resection for oral carcinomas and oral potentially malignant disorders, the authors analyzed the color of unstained lesions (USLs) in relation to histopathological findings. After vital iodine staining, USLs were seen in 48 of 54 patients (88.9%). A significant difference was seen in the value of lightness between stained lesions (SLs) and USLs for patients with moderate and severe epithelial dysplasia ( P < 0.001). The deviation between the macroscopically observable and the histopathological boundaries was −0.65 ± 1.26 mm (range: −4.36 to 1.52). Color charts prepared on the basis of values for lightness and hue reproduced the macroscopic color differences in USLs, suggesting that it may become possible to diagnose USLs histologically on the basis of the measured color values and use of color charts to help determine the resection area in surgery.

Oral cancer, especially oral squamous cell carcinoma (OSCC) is the most common head and neck cancer and is found in 270,000 patients annually worldwide . The American Cancer Society has set a goal of reducing the morbidity of oral cancer by 25% in the US between 1992 and 2015 and has shown a drop from 12.0% to 10.7% (mean change per year: −0.9%) in its interim report , but the 5-year survival rate has not improved in more than two decades . In the case of malignant tumors, early detection and early treatment are important to improve the survival rates of patients with oral cancer.

Vital iodine staining can be used immediately before resection as a useful and minimally invasive test for determining the area of resection for esophageal cancer . In this staining technique, iodine binds to glycogen granules in the cytoplasm of squamous cells, and this reaction results in a black–brown color. Many studies have shown that glycolysis is elevated in cancer cells , and such cellular areas with elevated glycolysis are generally seen as unstained lesions (USLs) during vital iodine staining. Since vital iodine staining was first performed on the cervix and esophageal mucosa , it has been combined routinely with endoscopy of the upper gastrointestinal tract, and it has contributed markedly to the early detection of esophageal cancer .

The oral mucosa, the lining of which is made up of non-keratinizing stratified squamous epithelium (buccal mucosa, oral vestibule, ventral surface of the tongue [lateral margin to the inferior surface], floor of the mouth and soft palate) is stained with iodine; but the masticatory mucosa (attached gingiva and hard palate) and specialized mucosa (dorsal lingual mucosa) are not . Compared with the upper gastrointestinal tract, the thickness of the mucosal epithelium varies in the oral cavity (100–500 μm), and the presence of teeth, saliva and movement of the tongue make it difficult to observe tissue samples under similar conditions.

The objectives of the present study were to analyze the color of USLs in the vicinity of oral carcinomas and oral potentially malignant disorders (OPMD) in relation to histopathological findings, and to determine whether the color measurement provided efficient criteria for determining the resection area for treatment of these cancers.

Patients and methods

Fifty-four patients who had conditions diagnosed as OSCC or OPMD and who underwent biopsy or surgery under general anesthesia at the Tokyo Medical and Dental University Hospital Faculty of Dentistry from July 2007 to September 2008 were included in this study. The inclusion criteria were: OSCC patients classified in the T1 or T2 category; OPMD patients who underwent biopsy for definite diagnosis or were selected for surgical treatment; patients whose lesions were measureable and could be illuminated by an artificial sunlight lamp. The mean age of the patients was 60.7 years (range 25–83 years), and their clinicopathological features are shown in Table 1 . This study was approved by the Ethics Review Board of the Faculty of Dentistry (NO.277). The study was explained to all patients and informed consent was obtained from them. Clinical staging was performed on the basis of the International Union Against Cancer (UICC) , and oral leukoplakia was classified according to WHO criteria .

Table 1
Clinicopathological features of patients (n = 54).
Characteristic No.
Age (years)
<60 23
≧60 31
Sex
Male 32
Female 22
Site of disease
Tongue 46
Buccal mucosa 4
Soft Palate 3
Oral Vestibule 1
Histological diagnosis
OSCC a 38
OL b 15
Epithelial hyperplasia 1
TNM classification of OSCC
T category
T1 20
T2 18
N category
N0 35
N1 3
Classification of OL
homogeneous 11
nonhomogeneous 4

a OSCC, oral squamous cell carcinoma.

b OL, oral leukoplakia.

Staining solutions and procedures

A 3% Lugol’s solution containing 3 g of iodine, 6 g of potassium iodine, and 100 ml of distilled water was used for staining according to the following procedure. First, the untreated lesion was photographed, and the colorimeter (CS-100, Konica Minolta Holdings, Inc., Tokyo, Japan) was calibrated in the oral cavity. The 3% Lugol’s solution was applied, and the area was rinsed with water and dried. Color measurement was performed after 2 min, and the USLs were photographed. Sodium thiosulfate was applied. USLs were defined as uncolored areas that appeared after iodine staining, excluding macroscopically observable white lesions and tumorous areas. Stained lesions (SLs) were defined as colored normal mucosa that appeared proximate to USLs.

Staining solutions and procedures

A 3% Lugol’s solution containing 3 g of iodine, 6 g of potassium iodine, and 100 ml of distilled water was used for staining according to the following procedure. First, the untreated lesion was photographed, and the colorimeter (CS-100, Konica Minolta Holdings, Inc., Tokyo, Japan) was calibrated in the oral cavity. The 3% Lugol’s solution was applied, and the area was rinsed with water and dried. Color measurement was performed after 2 min, and the USLs were photographed. Sodium thiosulfate was applied. USLs were defined as uncolored areas that appeared after iodine staining, excluding macroscopically observable white lesions and tumorous areas. Stained lesions (SLs) were defined as colored normal mucosa that appeared proximate to USLs.

Tissue processing and evaluation

A shallow incision was made using a scalpel in 34 patients who had a clear, straight border between the unstained and stained mucosa ( Fig. 1 A) . After fixing in 10% buffered formalin, 4-μm sections were stained using hematoxylin-eosin (H-E) in all cases. For histopathological evaluation of USLs, epithelial dysplasia was classified as mild, moderate or severe according to WHO standards. If atypical findings were not observed, hyperkeratosis and acanthosis of the simple elongated epithelial layer were grouped into epithelial hyperplasia. Periodic acid Schiff (PAS) staining and PAS staining following enzymatic digestion with 0.1% α-amylase were performed. A histopathological diagnosis was made ( Fig. 1 B) on the basis of these stained sections. To evaluate the boundaries between USLs and SLs, the number of PAS-positive cells in the non-keratinizing epithelial layer was counted over a 25 mm 2 area for USLs/SLs and surgical margin areas, which were defined as tissue within 5 mm from the boundary line and the adjacent surgical margin, respectively, using an eyepiece micrometer (U-OCMSQ10/10, Olympus, Inc., Tokyo, Japan) at a magnification of × 200. The percentage of PAS-positive cells was calculated. The authors defined the deviation as the distance between the macroscopically observable boundary and the histological boundary, measured as the distance from the superficial incision to the normal epithelial layer ( Fig. 1 C,D). The status of all resection margins was examined in permanent paraffin sections.

Fig. 1
Processing and evaluation of tissue. (A) Specimen after resection (Patient No. 44: oral squamous cell carcinoma; T2N0M0). Circle and black line indicate color measuring point for USLs and cut mark of boundary line between SLs and USLs, respectively. (B) Histopathological diagnosis corresponding to sections of specimen. Arrows indicate surgical margin area defined as the tissue surrounding the adjacent marginal area. Yellow line, mild epithelial dysplasia; green line, moderate epithelial dysplasia; pink line, severe epithelial dysplasia; red cross, cancerous lesions; black line, cut mark of boundary line between SLs and USLs; white circle, color measuring point for USLs. (C) Arrow indicates shallow incision in the specimen showing macroscopic boundary line between SLs and USLs (H-E staining ×40). (D) Schema for measurement of deviation. The deviation was defined as the distance between the macroscopic and histopathological boundary lines. The histopathological border in the specimen is indicated by the zero point.

Color measurement

Color was measured using a colorimeter (CS-100, Konica Minolta Holdings, Inc., Japan) according to the method of Yamashiro as follows: an artificial sunlight lamp (SOLAX XC-100, Seric Ltd., Tokyo, Japan) was used to illuminate the lesion at a 45° angle and a distance of 1 m, and color was measured in the vertical direction. A close-up lens No.122 (measurement diameter 3.2–4.3 mm) was attached to the colorimeter and color was measured within the wavelength range of 323–368 mm. Numerical values were recorded using the data processor DP-101 (Konica Minolta Holdings, Inc., Japan). Measuring points of USLs and SLs were defined as the area within 10 mm from the boundary line. In each test, five measurements were taken to eliminate marked variations and an average value was calculated. D55 was used as the light source and data were calculated in the Y, x, y mode. Using a dental mirror-type standard white board, an arbitrary standard calibration was carried out under the same conditions as the test areas. For sanitation, fingertips cut from disposable, sterilized gloves were used to cover the areas and were changed for each patient.

The CIE L * a * b * color system recommended by the Commission Internationale dl’Eclairage in 1976 is used to describe the developed color. With this system, L * indicates lightness and a * and b * indicate hue. The following formulas are used for converting Y, x, y into L *, a *, b * .

L=116(YYn)1/316L=116(YYn)1/316
L = 116 Y Y n 1 / 3 − 16
a=500((XXn)1/3(YYn)1/3)
a = 500 X X n 1 / 3 − Y Y n 1 / 3
b=200((YYn)1/3(ZZn)1/3)
b = 200 Y Y n 1 / 3 − Z Z n 1 / 3
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Feb 8, 2018 | Posted by in Oral and Maxillofacial Surgery | Comments Off on Colorimetric analysis of unstained lesions surrounding oral squamous cell carcinomas and oral potentially malignant disorders using iodine
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