Review Article
Chemiluminescence: A diagnostic adjunct in oral precancer and cancer: A review ABSTRACT Oral cancer is one of the most common cancers in India. It’s also associated with poor survival rate. Early diagnosis is the only way of reducing the high morbidity and mortality associated with it. However, most often there is delay in its diagnosis. Several adjuncts have been developed to aid in the diagnosis of cancer in its pre‑cancerous and early stage. Chemiluminescence is one of the newly developed adjuncts, which is still in its infancy. This article reviews chemiluminescence technique and its applications in oral cancer diagnosis. KEY WORDS: Chemiluminescence, diagnosis, oral cancer, toluidine blue, Vizilite
INTRODUCTION Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality world‑wide.[1] In India, oral cancer is the most prevalent cancer in men and the third most prevalent cancer in women and it makes up 40% of all cancers in the country.[1] Despite numerous advances in the treatment, the 5 year survival has remained approximately 50% for the last 50 years. The prognosis for the patients with OSCC that is treated early is much better, with 5 year survival rates as high as 80% and in addition, quality of life improves after early treatment.[2] Numerous adjunctive techniques have been developed with the aim of diagnosing cancer in its premalignant and early stage. Some of these techniques are cytological methods, vital tissue staining, histological, molecular and optical techniques.[2,3] Exfoliative cytology is one of the commonly used diagnostic aid. However, relative superficial samples, samples admixed with blood and necrotic cells reduces its diagnostic ability.[4] The new modified cytological techniques such as oral brush biopsy and liquid based cytology and other advanced molecular techniques are been studied but are comparatively expensive.[2‑4] Vital tissue staining though is simple and economical, its specificity is low.[5,6] Biopsy is considered the gold standard, but is invasive and may be non‑diagnostic in early stages.[3,4] Optical techniques are in the recent forefront. They are based on the ability of the tissues to fluoresce. The different optical techniques include fluorescence spectroscopy, Raman spectroscopy, elastic scattering spectroscopy, chemiluminescence (Vizilite) and
velscope.[7] Chemiluminescence, like the other optical techniques has been designed to use in early stages of cancer when the changes have occurred at the molecular level with or without the minimal clinical expression. This article reviews the technique and applications of chemiluminescence to oral oncology. CHEMILUMINESCENCE Luminescence in living organisms (bioluminescence) has been reported as far back as 1500 BC in the Chinese literature, the best‑known examples being emission of light from fireflies and glow‑worms. The first report of artificial luminescence (chemiluminescence) was in 1669 by a German physician, Henning Brand, who discovered phosphorus. The term “chemiluminescence” was first coined by Eilhardt Weidemann in 1888.[8,9] Luminescence is a term used to describe the emission of light, which occurs when a molecule in an excited state relaxes to its ground state. The various types of luminescence differ in the source of energy to obtain the excited state. This energy can be supplied by electromagnetic radiation (photoluminescence also termed as fluorescence or phosphorescence), by heat (pyroluminescence), by frictional forces (triboluminescence), by electron impact (cathodoluminescence) or by crystallization (crystalloluminescence). In chemiluminescence, the energy is produced by a chemical reaction.[10] This phenomenon is observed when the vibronically excited product of an exogenic reaction relaxes to its ground state with emission of photons. The chemical reaction
Journal of Cancer Research and Therapeutics - July-September 2014 - Volume 10 - Issue 3
Raju Shashidara, Huchanahalli Sheshanna Sreeshyla1, Udyavara Sridhara Sudheendra Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet, 1 Department of Oral Pathology and Microbiology, JSS Dental College and Hospital (A Constituent of JSS University), Mysore, Karnataka, India For correspondence: Dr. Huchanahalli Sheshanna Sreeshyla, Hampi Circle, Vijaynagara, Mysore ‑ 570 017, Karnataka, India. E‑mail: sreeshylahs@ gmail.com
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.138215 PMID: *** Quick Response Code:
487
Shashidara, et al.: Chemiluminescence in cancer diagnosis
produces energy in sufficient amount to induce the transition of an electron from its ground state to an excited electronic state. This electronic transition is often accompanied by vibrational and rotational changes in the molecule.[8‑10] Chemiluminescent reactions emit light of varying degrees of intensity and lifetime, with colors that span the visible spectrum. There are many systems of chemiluminescence of which the two most widely used are the luminol based and the peroxy‑oxalate based systems.[8,9] Chemiluminescence has been employed in the field of obstetrics and gynecology for many years as an adjunct for the early detection of cervical cancer and pre‑cancer. The technique is referred to as speculoscopy and it involves inspection of the cervix following the application of 5% acetic acid with chemiluminescent light based on the peroxy‑oxalate system.[2,8,11,12] This technique has been recently introduced to oral oncology for the detection of oral pre‑cancer and cancer, where it is referred to as Vizilite or lumenoscopy. The ViziLite ® (Zila Pharmaceuticals, Phoenix, AZ) was approved as adjunctive technology by U.S. Food and Drug Administration (FDA) in November 2002.[7,13,14] Vizilite was given a dental reimbursement code by the Code Revision Committee of the ADA in June 2004.[14] The Vizilite‑Blue Oral Lesion Identification and Marking system, the Vizilite plus was approved by FDA in November 2004.[15] Vizilite Plus is the modified technique that consists of Vizilite and the Toluidine Blue Oral Lesion Marking System. It combines chemiluminescent technology to improve the visualization of oral lesions with Toluidine Blue Marking system.[16] Certain medical devices may be marketed without FDA approval, if the manufacturer claims that such device is “substantially equivalent” to another medical device that was sold before 1976, the FDA may grant a 510(k) clearance that allows the manufacturer to market that device without substantive review of its safety and efficacy. The Vizilite Plus with Toluidine Blue system is been given 510(k) clearance based on the manufacturer’s claim that the device was “substantially equivalent” to colposcopy examination lights used during a gynecologic examination.[1] Chemiluminescence for use in oral cavity is marketed under the names-Vizilite, Vizilite Plus and MicroLux DL. While the Vizilite Plus uses a disposable chemiluminescenct light packet, the MicroLux unit offers a reusable, battery powered light source. Several studies have examined chemiluminescence as an oral cancer screening aid. Although a single study has been published with MicroLux DL, the similarities in the emission characteristics between the two technologies make it unlikely that their results would be significantly different.[2,17] COMPONENTS The Vizilite kit is a single use product and it consists of 1% acetic acid solution, a capsule which emits light and a retractor (sheath and handle).[8,13] The Vizilite capsule or 488
chemiluminescent light stick consists of an outer shell of flexible plastic cap containing aspirin or acetyl salicylic acid and an inner fragile glass vial containing hydrogen peroxide. Activation of the capsule is achieved by flexing it, wherein, the inner fragile glass vial breaks releasing the hydrogen peroxide. The chemicals in the outer and inner compartments react to produce light of the bluish‑white color with a wavelength ranging from 430 to 580 nm. The light lasts for approximately 10 min.[3,8,13] The contents of the Vizilite 1% acetic acid solution are purified water, acetic acid, sodium benzoate, raspberry flavor, base of propylene glycol and alcohol.[8] Vizilite Plus consists of three swab components: Two swabs of 1% acetic acid rinse, including a post‑dye decolorizer and one swab with a metachromatic vital tissue dye, tolonium chloride solution (toluidine blue). The toluidine blue produces a deep blue color that aids in the easy visualization and delineation of the chemiluminescent positive area.[15,16] MECHANISM OF ACTION This technique detects the metabolic and structural changes in the mucosal tissues that by their nature have different absorbance and reflectance properties when exposed to various forms of light sources.[12] The acetic acid solution acts as cytoplasmic dehydration agent. It putatively removes debris and disrupts the glycoprotein barrier on the surface of the epithelium allowing penetration of the light.[2,3,14,16] The cytoplasmic dehydration changes its light refractile properties such that when chemiluminescent light is applied, the blue white light is absorbed by cells of the normal mucosa and is reflected by cells with abnormal nuclei including dysplastic and neoplastic cells, making the dysplastic and neoplastic epithelium appear as “white” hence the term “acetowhite”. Thus the normal epithelium takes on a blue hue, while the “acetowhite” lesions appear distinctly white.[3,14,16] The two chemicals in the Vizilite capsule, i.e. acetyl salicylic acid and hydrogen peroxide react to release energy. A fluorescent dye accepts this energy and converts it into light. The color of the fluorescent dye determines the resulting color of the light stick when the chemical solutions are mixed. The basic premise of the reaction is that the reaction between the two chemicals releases enough energy to excite the electrons in the fluorescent dye. This causes the electrons to jump to a higher energy level and then fall back down and release light. Specifically, the hydrogen peroxide oxidizes the phenyl oxalate ester, to form phenol and an unstable peroxyacid ester. The unstable peroxyacid ester decomposes, resulting in phenol and a cyclic peroxy compound. The cyclic peroxy compound decomposes to carbon dioxide. This decomposition reaction releases the energy that excites the dye. The detected signals may be related to the altered thickness of the epithelium, or to the presence of a higher density of nuclear content and mitochondrial matrix that preferentially reflect light. Hyperkeratinized or dysplastic lesions appear distinctly white when viewed under a diffuse low‑energy
Journal of Cancer Research and Therapeutics - July-September 2014 - Volume 10 - Issue 3
Shashidara, et al.: Chemiluminescence in cancer diagnosis
wavelength light.[7] Vizilite plus uses two technologies-to visualize and then to mark suspicious lesions in the oral cavity. Toluidine blue, the marking system used in this, is an acidophilic dye that selectively stains acidic tissue components such as deoxyribonucleic acid and ribonucleic acid. Due to the increased nuclear density, wider intracellular canals which enhances penetration of the dye, loss of cell cohesion and increased mitosis, the dysplastic and anaplastic cells are stained with toluidine blue and the normal epithelium remains unstained.[5,8,16,18] Chemiluminescence is used as an adjunct to the convectional oral mucosal examination to improve identification, evaluation and monitoring of oral lesions with increased risk for development of cancer. The other uses of this technology include immunoassays, gene probe assays, measurement of important enzymes and metabolites, liquid chromatography, pharmaceutical analysis and chemiluminescence imaging.[2,9] SCREENING PROCEDURE USING VIZILITE Conventional examination of the oral cavity using dental chair light.[8,12] • Record the nature and characteristics of the lesion if any detected during conventional examination[8,12] • Photograph the lesion[8,12] • Rinse mouth with 30 ml of 1% acetic acid and expectorate after 1 min. This removes the glycoprotein barrier and slightly dries the mucosa[8,12,13,18] • Activate and assemble the Vizilite capsule. This is done by bending the flexible outer capsule such that the inner brittle vial breaks[12,18] • Shake the capsule to mix the contents and then place it in the Vizilite retractor[8,18] • Dim ambient lights in the room and examine the oral cavity[8,13,18] • While normal epithelium appears blue, the altered epithelium appears acetowhite[13,18] • For toluidine blue marking system, rinse mouth with 10 ml of 1% tolonium chloride and expectorate after 1 min followed by rinse with 10 ml of 1% acetic acid and expectorate after 20 s[16,18] • Record and photograph the findings, if any[8] • Rinse mouth with water and expectorate after 20 s.[8] This technique has the advantage of easy to use, safe, non‑toxic to biological tissues, non‑invasive, chair side test and due to its single use the risk of cross contamination is reduced, provides real‑time results, easy to learn the technique and limited operator variability.[8,12,13,18] However, this technique also has several disadvantages such as it is expensive, needs dark environment, inability to indicate the appropriate site for biopsy and can be used only once for each patient, the sensitivity, specificity and effectiveness of the technique still remains undetermined and inability to objectively measure the visualization results.[12,13]
DISCUSSION A pilot study by Heber et al. provided a supportive evidence to the hypothesis that oral soft‑tissues exhibit features similar to the cervical epithelium following an acetic acid wash and visual inspection under chemiluminescent illumination.[6,12,18,19] This has prompted the use of Vizilite technique in the diagnosis of pre‑cancer and cancerous lesions of the oral cavity. Properties of chemiluminescent light, which makes it useful for detection of dysplasia are the multichromatic low intensity light with peak intensity
Chemiluminescence: A diagnostic adjunct in oral precancer and cancer: A review ABSTRACT Oral cancer is one of the most common cancers in India. It’s also associated with poor survival rate. Early diagnosis is the only way of reducing the high morbidity and mortality associated with it. However, most often there is delay in its diagnosis. Several adjuncts have been developed to aid in the diagnosis of cancer in its pre‑cancerous and early stage. Chemiluminescence is one of the newly developed adjuncts, which is still in its infancy. This article reviews chemiluminescence technique and its applications in oral cancer diagnosis. KEY WORDS: Chemiluminescence, diagnosis, oral cancer, toluidine blue, Vizilite
INTRODUCTION Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy and is a major cause of cancer morbidity and mortality world‑wide.[1] In India, oral cancer is the most prevalent cancer in men and the third most prevalent cancer in women and it makes up 40% of all cancers in the country.[1] Despite numerous advances in the treatment, the 5 year survival has remained approximately 50% for the last 50 years. The prognosis for the patients with OSCC that is treated early is much better, with 5 year survival rates as high as 80% and in addition, quality of life improves after early treatment.[2] Numerous adjunctive techniques have been developed with the aim of diagnosing cancer in its premalignant and early stage. Some of these techniques are cytological methods, vital tissue staining, histological, molecular and optical techniques.[2,3] Exfoliative cytology is one of the commonly used diagnostic aid. However, relative superficial samples, samples admixed with blood and necrotic cells reduces its diagnostic ability.[4] The new modified cytological techniques such as oral brush biopsy and liquid based cytology and other advanced molecular techniques are been studied but are comparatively expensive.[2‑4] Vital tissue staining though is simple and economical, its specificity is low.[5,6] Biopsy is considered the gold standard, but is invasive and may be non‑diagnostic in early stages.[3,4] Optical techniques are in the recent forefront. They are based on the ability of the tissues to fluoresce. The different optical techniques include fluorescence spectroscopy, Raman spectroscopy, elastic scattering spectroscopy, chemiluminescence (Vizilite) and
velscope.[7] Chemiluminescence, like the other optical techniques has been designed to use in early stages of cancer when the changes have occurred at the molecular level with or without the minimal clinical expression. This article reviews the technique and applications of chemiluminescence to oral oncology. CHEMILUMINESCENCE Luminescence in living organisms (bioluminescence) has been reported as far back as 1500 BC in the Chinese literature, the best‑known examples being emission of light from fireflies and glow‑worms. The first report of artificial luminescence (chemiluminescence) was in 1669 by a German physician, Henning Brand, who discovered phosphorus. The term “chemiluminescence” was first coined by Eilhardt Weidemann in 1888.[8,9] Luminescence is a term used to describe the emission of light, which occurs when a molecule in an excited state relaxes to its ground state. The various types of luminescence differ in the source of energy to obtain the excited state. This energy can be supplied by electromagnetic radiation (photoluminescence also termed as fluorescence or phosphorescence), by heat (pyroluminescence), by frictional forces (triboluminescence), by electron impact (cathodoluminescence) or by crystallization (crystalloluminescence). In chemiluminescence, the energy is produced by a chemical reaction.[10] This phenomenon is observed when the vibronically excited product of an exogenic reaction relaxes to its ground state with emission of photons. The chemical reaction
Journal of Cancer Research and Therapeutics - July-September 2014 - Volume 10 - Issue 3
Raju Shashidara, Huchanahalli Sheshanna Sreeshyla1, Udyavara Sridhara Sudheendra Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet, 1 Department of Oral Pathology and Microbiology, JSS Dental College and Hospital (A Constituent of JSS University), Mysore, Karnataka, India For correspondence: Dr. Huchanahalli Sheshanna Sreeshyla, Hampi Circle, Vijaynagara, Mysore ‑ 570 017, Karnataka, India. E‑mail: sreeshylahs@ gmail.com
Access this article online Website: www.cancerjournal.net DOI: 10.4103/0973-1482.138215 PMID: *** Quick Response Code:
487
Shashidara, et al.: Chemiluminescence in cancer diagnosis
produces energy in sufficient amount to induce the transition of an electron from its ground state to an excited electronic state. This electronic transition is often accompanied by vibrational and rotational changes in the molecule.[8‑10] Chemiluminescent reactions emit light of varying degrees of intensity and lifetime, with colors that span the visible spectrum. There are many systems of chemiluminescence of which the two most widely used are the luminol based and the peroxy‑oxalate based systems.[8,9] Chemiluminescence has been employed in the field of obstetrics and gynecology for many years as an adjunct for the early detection of cervical cancer and pre‑cancer. The technique is referred to as speculoscopy and it involves inspection of the cervix following the application of 5% acetic acid with chemiluminescent light based on the peroxy‑oxalate system.[2,8,11,12] This technique has been recently introduced to oral oncology for the detection of oral pre‑cancer and cancer, where it is referred to as Vizilite or lumenoscopy. The ViziLite ® (Zila Pharmaceuticals, Phoenix, AZ) was approved as adjunctive technology by U.S. Food and Drug Administration (FDA) in November 2002.[7,13,14] Vizilite was given a dental reimbursement code by the Code Revision Committee of the ADA in June 2004.[14] The Vizilite‑Blue Oral Lesion Identification and Marking system, the Vizilite plus was approved by FDA in November 2004.[15] Vizilite Plus is the modified technique that consists of Vizilite and the Toluidine Blue Oral Lesion Marking System. It combines chemiluminescent technology to improve the visualization of oral lesions with Toluidine Blue Marking system.[16] Certain medical devices may be marketed without FDA approval, if the manufacturer claims that such device is “substantially equivalent” to another medical device that was sold before 1976, the FDA may grant a 510(k) clearance that allows the manufacturer to market that device without substantive review of its safety and efficacy. The Vizilite Plus with Toluidine Blue system is been given 510(k) clearance based on the manufacturer’s claim that the device was “substantially equivalent” to colposcopy examination lights used during a gynecologic examination.[1] Chemiluminescence for use in oral cavity is marketed under the names-Vizilite, Vizilite Plus and MicroLux DL. While the Vizilite Plus uses a disposable chemiluminescenct light packet, the MicroLux unit offers a reusable, battery powered light source. Several studies have examined chemiluminescence as an oral cancer screening aid. Although a single study has been published with MicroLux DL, the similarities in the emission characteristics between the two technologies make it unlikely that their results would be significantly different.[2,17] COMPONENTS The Vizilite kit is a single use product and it consists of 1% acetic acid solution, a capsule which emits light and a retractor (sheath and handle).[8,13] The Vizilite capsule or 488
chemiluminescent light stick consists of an outer shell of flexible plastic cap containing aspirin or acetyl salicylic acid and an inner fragile glass vial containing hydrogen peroxide. Activation of the capsule is achieved by flexing it, wherein, the inner fragile glass vial breaks releasing the hydrogen peroxide. The chemicals in the outer and inner compartments react to produce light of the bluish‑white color with a wavelength ranging from 430 to 580 nm. The light lasts for approximately 10 min.[3,8,13] The contents of the Vizilite 1% acetic acid solution are purified water, acetic acid, sodium benzoate, raspberry flavor, base of propylene glycol and alcohol.[8] Vizilite Plus consists of three swab components: Two swabs of 1% acetic acid rinse, including a post‑dye decolorizer and one swab with a metachromatic vital tissue dye, tolonium chloride solution (toluidine blue). The toluidine blue produces a deep blue color that aids in the easy visualization and delineation of the chemiluminescent positive area.[15,16] MECHANISM OF ACTION This technique detects the metabolic and structural changes in the mucosal tissues that by their nature have different absorbance and reflectance properties when exposed to various forms of light sources.[12] The acetic acid solution acts as cytoplasmic dehydration agent. It putatively removes debris and disrupts the glycoprotein barrier on the surface of the epithelium allowing penetration of the light.[2,3,14,16] The cytoplasmic dehydration changes its light refractile properties such that when chemiluminescent light is applied, the blue white light is absorbed by cells of the normal mucosa and is reflected by cells with abnormal nuclei including dysplastic and neoplastic cells, making the dysplastic and neoplastic epithelium appear as “white” hence the term “acetowhite”. Thus the normal epithelium takes on a blue hue, while the “acetowhite” lesions appear distinctly white.[3,14,16] The two chemicals in the Vizilite capsule, i.e. acetyl salicylic acid and hydrogen peroxide react to release energy. A fluorescent dye accepts this energy and converts it into light. The color of the fluorescent dye determines the resulting color of the light stick when the chemical solutions are mixed. The basic premise of the reaction is that the reaction between the two chemicals releases enough energy to excite the electrons in the fluorescent dye. This causes the electrons to jump to a higher energy level and then fall back down and release light. Specifically, the hydrogen peroxide oxidizes the phenyl oxalate ester, to form phenol and an unstable peroxyacid ester. The unstable peroxyacid ester decomposes, resulting in phenol and a cyclic peroxy compound. The cyclic peroxy compound decomposes to carbon dioxide. This decomposition reaction releases the energy that excites the dye. The detected signals may be related to the altered thickness of the epithelium, or to the presence of a higher density of nuclear content and mitochondrial matrix that preferentially reflect light. Hyperkeratinized or dysplastic lesions appear distinctly white when viewed under a diffuse low‑energy
Journal of Cancer Research and Therapeutics - July-September 2014 - Volume 10 - Issue 3
Shashidara, et al.: Chemiluminescence in cancer diagnosis
wavelength light.[7] Vizilite plus uses two technologies-to visualize and then to mark suspicious lesions in the oral cavity. Toluidine blue, the marking system used in this, is an acidophilic dye that selectively stains acidic tissue components such as deoxyribonucleic acid and ribonucleic acid. Due to the increased nuclear density, wider intracellular canals which enhances penetration of the dye, loss of cell cohesion and increased mitosis, the dysplastic and anaplastic cells are stained with toluidine blue and the normal epithelium remains unstained.[5,8,16,18] Chemiluminescence is used as an adjunct to the convectional oral mucosal examination to improve identification, evaluation and monitoring of oral lesions with increased risk for development of cancer. The other uses of this technology include immunoassays, gene probe assays, measurement of important enzymes and metabolites, liquid chromatography, pharmaceutical analysis and chemiluminescence imaging.[2,9] SCREENING PROCEDURE USING VIZILITE Conventional examination of the oral cavity using dental chair light.[8,12] • Record the nature and characteristics of the lesion if any detected during conventional examination[8,12] • Photograph the lesion[8,12] • Rinse mouth with 30 ml of 1% acetic acid and expectorate after 1 min. This removes the glycoprotein barrier and slightly dries the mucosa[8,12,13,18] • Activate and assemble the Vizilite capsule. This is done by bending the flexible outer capsule such that the inner brittle vial breaks[12,18] • Shake the capsule to mix the contents and then place it in the Vizilite retractor[8,18] • Dim ambient lights in the room and examine the oral cavity[8,13,18] • While normal epithelium appears blue, the altered epithelium appears acetowhite[13,18] • For toluidine blue marking system, rinse mouth with 10 ml of 1% tolonium chloride and expectorate after 1 min followed by rinse with 10 ml of 1% acetic acid and expectorate after 20 s[16,18] • Record and photograph the findings, if any[8] • Rinse mouth with water and expectorate after 20 s.[8] This technique has the advantage of easy to use, safe, non‑toxic to biological tissues, non‑invasive, chair side test and due to its single use the risk of cross contamination is reduced, provides real‑time results, easy to learn the technique and limited operator variability.[8,12,13,18] However, this technique also has several disadvantages such as it is expensive, needs dark environment, inability to indicate the appropriate site for biopsy and can be used only once for each patient, the sensitivity, specificity and effectiveness of the technique still remains undetermined and inability to objectively measure the visualization results.[12,13]
DISCUSSION A pilot study by Heber et al. provided a supportive evidence to the hypothesis that oral soft‑tissues exhibit features similar to the cervical epithelium following an acetic acid wash and visual inspection under chemiluminescent illumination.[6,12,18,19] This has prompted the use of Vizilite technique in the diagnosis of pre‑cancer and cancerous lesions of the oral cavity. Properties of chemiluminescent light, which makes it useful for detection of dysplasia are the multichromatic low intensity light with peak intensity
