Salivary Lactate Dehydrogenase (LDH) As a Marker for Radiation-Induced Mucositis in Head and Neck Cancers: a Preliminary Study
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Archive of SID Original Article Middle East Journal of Cancer; AOpcrtiol b2e0r1 290; 180;( 29)(:4 1):0 238-181-0294 Salivary Lactate Dehydrogenase (LDH) as a Marker for Radiation-induced Mucositis in Head and Neck Cancers: A Preliminary Study Arnadi R Shivashankara*, Raees Tonse**, Sucharitha Suresh***, Thomas George***, Mamidipudi Srinivasa Vidyasagar**, Suresh Rao****, Manjeshwar Shrinath Baliga**** ♦ *Departments of Biochemistry Father Muller Medical College, Mangalore, Karnataka, India **Radiation Oncology Father Muller Medical College, Mangalore, Karnataka, India ***Community Medicine, MBBS student, Father Muller Medical College, Mangalore, Karnataka, India ****Radiation Oncology, Mangalore Institute of Oncology, Pumpwell, Mangalore, Karnataka, India Abstract Background: In this study, we sought to understand the usefulness of salivary lactate dehydrogenase as a predictive marker for the development of radiation-induced mucositis. Methods: This was a prospective study with head and neck cancer patients who required curative radiotherapy (>60Gy). We collected patients’ saliva before the onset of radiation and after 2 Gy of radiation to assess lactate dehydrogenase levels. The patients received the stipulated oral and dental care. Data on incidence and severity of mucositis was collected using a preform sheet and oral mucositis assessment scale published by the Radiation Therapy Oncology Group throughout the 7-week treatment period. Results: Salivary lactate dehydrogenase increased with exposure to radiation (P<0.0001) and there was an observed association with mucositis severity ( P<0.0001; r = 0.515). Conclusion: The present results have established, for the first time, that salivary lactate dehydrogenase could be a useful predictive marker to understand the development of radiation-induced mucositis in patients with head and neck cancer. The proximity of the oral cavity for regular observation and saliva collection is an added advantage. ♦Corresponding Author: Keywords: Head and neck cancer, Salivary lactate dehydrogenase, Mucos itis ♦CMoarrnejessphownadri nSgh rAinuaththo rB: aliga, EPhlhD am Aghili, MD DReespeaarrtcmh eMnta nogfa loPrea tIhnosltoitguyte , Sofc hoOoln coof lMogeyd,i cinPeu,m Ispfwahealln , UMnainvgerasliotyr eo,f KMaerdniactaal kSac,i eInncdeisa , I5s7f5ah0a0n2 , Iran Introduction need radiation therapy during the Tel: +919-899914352422622946417 1 EmEmaila:i al:g mhislib_aellihgaam@@gmyaahiol.oc.ocmom Reports suggest that nearly 60% course of their treatment. However, of all people afflicted with cancers of the use of radiation is associated with the head and neck (H&N) region side effects and the development of Received: February 03, 2018; Accepted: September 08, 2018 www.SID.ir Archive of SID Salivary LDH as Marker for Radiation-induced Mucositis mucositis, which is characterized by inflammation environment. 8 Reports suggest that changes occur and pain of the mucosal surface membrane in the to salivary LDH levels in various oral radiosensitive oral mucosa that necessitates pathogeneses like gingivitis, periodontitis, and continuous medical care. 2-4 Oral mucositis is cancer. Hence, LDH is a useful marker in classified as tolerable (grades 1 and 2) and ascertaining the oral health of the individual. 10-15 intolerable (grades 3 and 4) depending on the Because ionizing radiation is cytotoxic, we severity. 3 While tolerable mucositis is clinically hypothesize that increased levels of LDH will be manageable, intolerable mucositis impairs diet, observed in cases with increased severity of affects quality of life, increases morbidity, and loss mucositis. We have conducted this study to of weight. 2-4 ascertain the role of salivary LDH in predicting From a cellular perspective, mucositis affects radiation-induced mucositis in H&N cancer the integrity of the mucosal barrier. It increases the patients who undergo curative cisplatin based chances for microbial growth and may lead to chemo-irradiation. systemic infections that are difficult to treat in immunocompromised cancer patients. 2-4 The Materials and Methods development of mucositis is a very complex We conducted this study from January 2012 to process. Studies have shown that the incidence and July 2013 in the Departments of Radiation severity of mucositis depend on patient related Oncology and Biochemistry at Father Muller factors like age, gender, body mass index, alcohol Medical College, Mangalore, Karnataka, India. and tobacco use, oral hygiene, stage of disease, The subjects consisted of histopathologically white blood cell count, salivary rate, and normal confirmed adult H&N cancer patients scheduled flora of the mouth in addition to extrinsic factors to receive curative chemoradiotherapy (60-70 like dose per fraction, total dose, overall treatment Gy). Table 1 lists the exclusion and inclusion duration, use of chemo-irradiation, and criteria. The Institutional Ethics Committee fractionation schedule. 5,6 However, none of these approved this study. factors have a predictive value and cannot predict if the patient will develop mucositis and, more Radiation treatment importantly, its severity during the course of the The participants of the study were scheduled radiation treatment. to receive external irradiation at an average energy Research in the area of diagnostics suggests that level of 6 MeV from a linear accelerator (Varian the biochemical endpoint from the site of treatment Medical Systems, Unique 2012, Palo Alto, CA, and/or body fluid is beneficial. 7 As H&N cancers USA). Planned treatment included a curative pertain to the early part of the digestive tract and target dose of 60-70 Gy, 5 days per week without encompass the easily accessible oral cavity, the any intended gap, and no more than one fraction saliva produced in this region is a useful body per day of 2 Gy for 6-7 consecutive weeks. fluid, especially in ailments of the oral cavity. 8 Patients received their cisplatin infusion (50 2 16,17 Saliva collection does not need skilled personnel mg/m /day; IV) as per standard guidelines. or special equipment and is non-invasive. Lactate dehydrogenase (LDH), a crucial Saliva collection enzyme in metabolism, is involved in catalyzing During the first visit, we explained the nature the reversible conversion of pyruvate and lactate and purpose of the study to eligible patients who during glycolysis and gluconeogenesis. 9 Lactate satisfied the inclusion criteria. The explanations dehydrogenase is present in the cell cytoplasm in were provided in either English or the patients’ normal healthy conditions. However, exposure mother tongue (Kannada, Tulu or Malayalam) of the cell to a cytotoxic agent leads to cell rupture by one of the investigators. The subjects were and release of LDH into the extracellular informed that they had the right to withdraw from www.SID.ir Middle East J Cancer 2019; 10(2): 103-110 104 ArchiveArnadi R S hofivas hSIDankara et al. the study at any time during the study and non- spectrophotometer (Shimadzu, Japan). The LDH willingness to participant in the study would not assay was performed according to the kinetic deprive them of the necessary planned treatment. spectrophotometric method described by The patients who consented were then included Demetriouet al. 19 using a reagent kit obtained in the study after they provided a written informed from Roche Diagnostics. The assay was based on consent to participate. an LDH-catalyzed reduction of pyruvate with Unstimulated saliva was collected in NADH to form NAD+. The rate of oxidation of accordance with the method suggested by NADH to NAD+ was measured as a decrease in Navazesh at two time points: i) before the start of absorbance at 340 nm and expressed in terms of radiation treatment (day 0) before exposure to units/mg of protein. We used the internal and the first 2 Gy fraction and ii) 24 h after external quality control program from Biorad to administration of the first 2 Gy fraction and prior ensure accuracy and precision of the LDH values. to the second 2 Gy fraction. Each patient was asked to thoroughly rinse Clinical evaluation for mucositis the mouth area with distilled water to remove Mucositis grading any food debris. After 10 min, the patients were One of the investigators (RT) assessed the requested to salivate into a sterile plastic cup. degree of mucositis before the start of treatment Salivary flow rate (mL/min) was measured by and weekly during the radiation treatments in the following formula: 18 accordance with Radiation Therapy Oncology Group (RTOG) guidelines. The severity of oral Weight of container with saliva (g) -Weight of container without saliva (g) Duration of saliva collection mucositis was graded from 0 to 4 based on the RTOG guidelines. 3 Grades 0, 1 and 2 were The collected saliva was immediately ‘tolerable’ and grades 3 and 4 were ‘intolerable’ transported to the Biochemistry Laboratory in an forms of mucositis. 3 Calibration of assessment was ice box. The collected saliva was centrifuged at not required because only one researcher evaluated 3000 rpm for 10 minutes, and the supernatants the patients throughout the study period. were stored in a freezer (-20ºC). All patients were provided with the standard oral, dental, and medical supportive care. Patients LDH in saliva were provided with a 1:100 povidone-iodine The stored saliva was removed from the solution (1 mL of Betadine and 100 mL of water) freezer, thawed, and analyzed using appropriate as recommended by Madan and co-workers. 20 blanks, controls, and standards in a UV-visible Patients were requested to perform oral cleanings Figure 1. Incidence of different grades of mucositis during the 7- Figure 2. Average mucositis during the 7-week treatment course week treatment course for head and neck (H&N) cancers treated with for head and neck (H&N) cancers treated with radiation (>60 Gy). radiation (>60 Gy). RTOG: Radiation Therapy Oncology Group. RTOG: Radiation Therapy Oncology Group. www.SID.ir 105 Middle East J Cancer 2019; 10(2): 103-110 Archive of SID Salivary LDH as Marker for Radiation-induced Mucositis Table 1. Inclusion and exclusion criteria for study patient selection. Inclusion criteria Exclusion criteria 1. Age >18 years 1. Unwilling to be a part of the study. 2.