THESIS – SYNOPSIS
DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY
A.B.SHETTY MEMORIAL INSTITUTE OF DENTAL SCIENCES
DERLAKATTE, MANGALORE – 575018
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1. / NAME OF THE CANDIDATE (IN BLOCK LETTERS) AND ADDRESS / DR. CHETHANAPOST GRADUATE STUDENT
DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
A.B.SHETTY MEMORIAL INSTITUTE OF DENTAL SCIENCES,
DERLAKATTE, MANGALORE - 575018
2. / NAME OF THE INSTITUTION / A.B.SHETTY MEMORIAL INSTITUTE OF DENTAL SCIENCES
DERLAKATTE, MANGALORE - 575018
3. / COURSE OF THE STUDY AND SUBJECT / MASTER OF DENTAL SURGERY, DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY
4. / DATE OF ADMISSION TO COURSE / MAY 2008
5. TITLE OF THE TOPIC:
“QUALITATIVE AND QUANTITATIVE ANALYSIS OF ACUTE PHASE PROTEINS IN POST-CHEMO RADIATION MUCOSITIS”
6. / BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR THE STUDY:
Oral mucositis induced by radiation is a reactive inflammatory process of the oropharyngeal mucous membrane. The first sign of inflammatory changes in the mucosa are clinically seen by the end of the first week of a conventional radiation protocol. It is seen as a white discoloration or reddening (erythema) of the mucosa.1
During radiation therapy, varying stages of mucositis may develop, such as the formation of pseudomembranes (3 to 4week) and ulcerations. It is associated with significant morbidity; pain, odynodysphagia, dysgeusia, dehydration and malnutrition that reduces the quality of life of the affected patients.1
Increase in pro- inflammatory cytokines and acute phase proteins (APP) are associated with the development of mucositis and are likely to play important roles in mediating injury and in signaling pathways. The levels of pro- inflammatory cytokines like interleukin 1 beta (IL-β), interleukin 6 (IL-6) and tumor necrosis factor (TNF) increase before tissue damage is apparent.2
APP are a class of proteins whose phase concentrations fluctuate in response to inflammation. This response is called the acute phase reaction. These proteins are produced by the liver and include C – reactive protein (CRP), serum amyloid A, serum amyloid P, fibrinogen, ferritin.3
APP contribute to host defence, they directly neutralize inflammatory agents, help to minimize the extent of local tissue damage, as well as participate in tissue repair and regeneration.3
The best known of the APP is CRP, a protein that rises in the blood with inflammation. Most APP are induced between 50% and several folds over normal levels.3
Scoring oral mucositis provides a means of evaluating the relationship between dosage, mode of delivery and complications. It is also useful in assessing the impact of therapy on patient morbidity, mortality and the quality of life of patients. The most commonly used scale is the World Health Organization (WHO) classification which grades oral mucositis into 0, 1, 2, 3 and 4.4
Erythrocyte sedimentation rate (ESR) is an acute-phase reactant test that reacts to acute conditions in the body and is used to evaluate the health status of patients when an inflammatory, neoplastic or infectious disease is suspected.5
In this present study our aim is to correlate APP and ESR with radiation induced severity of mucositis in patients receiving radiation treatment and concurrent chemotherapy.
6.2 REVIEW OF LITERATURE:
Oral complications are an inevitable sequelae of head and neck radiation, involving pseudomebranous and ulcerative mucositis in about 80% of the patients. It is often painful, afflicts oral functioning such as swallowing and eating, diminishes the quality of life and can be dose- limiting.6
A study on patients with head and neck cancer undergoing radical radiation therapy reported that maximal levels of CRP and ESR occurred at the end of radiotherapy (7 weeks) and then decreased on a 1 month follow- up. Patients receiving CRT (radiation and concurrent chemotherapy) had statistically greater elevations in ESR reaching a peak level in the second week compared to radiation treatment alone patients.7
A study performed to analyse the role of Palifermin in controlling mucositis in dose- intense conventional polychemotherapy patients showed a peak rise in the level of CRP (25000mg/l) in the first month followed by a sudden decline in the subsequent months corresponding to the healing phase of mucositis.8
A similar biochemical study in multiple myeloma patients to analyse the CRP level in high dose mephalan and autologous transplants gave a highest mean CRP of 100mg/l and CRP velocity of 15mg/l/day. This was correlating with the acute phase of mucositis in the study group and peaked with the level of mucositis.9
In a study based on the assessment of APP in acute ischemic stroke, it was reported that CRP, complement 3 and fibrinogen reached their highest values on the 3rd day; ceruloplasmin and complement 4 on the 5th day and haptoglobin on the 10th day.10
A study on the response of APP to infection in severe malnutrition in children showed that with the exception of fibrinogen, the plasma concentration of CRP, α1 acid glycoprotein, α1 – antitrypsin, haptoglobin were significantly higher after 2 days postadmission when compared to that after 9 days and at recovery.11
Analysis of the role of serum APP as an indicator of liver failure after liver resection showed that post-operative liver failure correlated with higher negative and lower positive APP at baseline and lower negative and lower positive APP on post-operative day 3, 12 and 45.12
A relationship between inflammatory oral disease and CRP was reported. The highest incidence of positive CRP tests and the strongest CRP test reactions were observed in patients with acute alveolar abscesses.13
6.3 AIMS OF THE STUDY:
1. Qualitative assessment of oral mucositis in patients undergoing chemo- radiotherapy.
2. Assessment of ESR and acute phase protein (APP), C-reactive protein (CRP) and blood leukocyte levels as an indicator of inflammation in patients undergoing chemo- radiotherapy.
6.4 OBJECTIVES OF THE STUDY:
1. Clinical assessment and scoring of oral mucositis.
2. Qualitative assessment of CRP in the serum of study and control groups.
3. Quantitative assessment of CRP in the serum of study and control groups.
4. Quantitative assessment of ESR in the study and control groups.
5. Quantitative estimation of blood leukocytes in the study and control groups.
6. Correlation of levels of leukocytes and CRP in the study group.
7. Comparison of CRP and ESR as a function of dose of exposure and stage of mucositis in the study group.
7. / MATERIALS AND METHODS:
7.1 SOURCE OF THE DATA:
The study group will comprise of 30 patients who are diagnosed with head and neck malignancy and planned to undergo chemo- radiotherapy as a treatment option. The study set up is at the Department of Oncology, Father Muller’s Oncology Centre, Mangalore which is a tertiary referral centre.
Study group:
30 patients diagnosed with head and neck malignancy who are to undergo chemo- radiotherapy as the treatment option.
Control group:
30 normal healthy persons who are systemically well and not under any medication and without any adverse habits.
Inclusion criteria:
Patients who are diagnosed with malignancy who are to undergo chemo- radiotherapy as the treatment option.
Exclusion criteria:
1. Patients with any local and systemic illness, infection or inflammation.
2. Any medication other than chemo- radiotherapy.
3. Pregnancy
7.2 METHOD OF COLLECTION OF DATA:
The procedure involves clinical scoring and collection of blood samples from both study and control groups.
Study group:
It is planned to perform the above mentioned clinical procedures on the first day prior to the commencement of radiotherapy. The further samples will be collected on the 7th, 14th ,28th and 42nd day. If the patient experiences any complications like delayed healing, weekly samples will be collected till the oral mucosa returns to normal.
Control group:
A single sample will be collected from healthy controls as per the inclusion and exclusion criteria.
I. ORAL MUCOSITIS SCORING:
Patients who are undergoing radiotherapy will be clinically evaluated for mucositis and scoring will be done based on the WHO scale4.
§ Grade 0 – no change
§ Grade 1 – soreness/ erythema
§ Grade 2 – erythema/ ulcers/ can eat solids
§ Grade 3 – ulcers/ requires liquid diet only
§ Grade 4 – alimentation not possible
II. ESTIMATION OF ESR:
1. Take 0.4 ml of 3.8 g/dl sodium citrate in a test tube.
2. Add 2ml of blood to the anticoagulant solution.
3. Fill the westergren tube exactly upto the zero mark.
4. Place the tube upright in the stand. It should fit evenly into the groove of the stand.
5. Allow the tube to stand for exactly 1 hour.
6. After 1 hour, note the level to which the red cell column has fallen.
7. Report the result in terms of mm/hr after 1 hour.
PREPARATION OF SERUM:
1. Collect 2ml of blood in a sterile 10ml plain centrifuge tube.
2. Allow it to fully clot (10min).
3. Centrifuge at 1500rpm for 10min in a standard centrifuge.
4. Collect the supernatant (serum)
III. QUALITATIVE ASESSMENT OF C – REACTIVE PROTEIN:
1. Pipette out one drop of the test specimen (serum) on the glass slide using a disposable pipette.
2. Add a drop of RHELAX CRP latex reagent to the drop of test specimen on the slide.
3. Using a mixing stick, mix the test specimen and the RHELAX CRP latex reagent uniformly over the entire circle.
4. Observe for agglutination macroscopically at 2 minutes.
IV. QUANTITATIVE ASSESSMENT OF C – REACTIVE PROTEIN:
1. Using isotonic saline prepare serial dilutions of the test specimen positive in the qualitative method 1:2, 1:4, 1:8, 1:16 till the dilution for estimation is reached.
2. Pipette each dilution of the test specimen onto separate reaction circles.
3. Add a drop of RHELAX CRP latex reagent to the drop of test specimen on the slide.
4. Using a mixing stick, mix the test specimen and the RHELAX CRP latex reagent uniformly over the entire circle.
5. Observe for agglutination macroscopically at 2 minutes.
INTERPRETATION OF TEST REULTS:
QUALITATIVE METHOD OF CRP ESTIMATION:
Agglutination is a positive test result and indicates the presence of detectable levels of CRP in the test specimen.
No agglutination is a negative test result and indicates the absence of detectable levels of CRP in the test specimen.
QUANTITATIVE METHOD OF CRP ESTIMATION:
Agglutination in the highest serum dilution corresponds to the amount of CRP in mg/dl in the test specimen.
CRP (mg/dl) = S х D
Where S = sensitivity of the reagent (0.6 mg/dl)
D = highest dilution of serum showing agglutination
V. QUANTITATIVE ESTIMATION OF BLOOD LEUKOCYTES:
1. Draw blood into the WBC pipette upto 0.5 mark.
2. Draw the diluting fluid upto 11 mark. The dilution is 1:20.
3. Mix the blood and diluting fluid by rotating the pipette on the palm keeping it horizontal.
4. Discard the first 2 drops and then charge the Neubauer chamber.
5. Count the cells in all the 4 corners of the Neubauer chamber.
STATISTICAL TEST TO BE USED:
- Friedman’s test will be used for the statistical analysis.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR INTERVENTIONS TO BE CONDUCTED ON PATIENTS OR OTHER HUMANS OR ANIMALS? [IF SO, PLEASE DESCRIBE BRIEFLY.]
Yes, blood samples will be collected from the patients after obtaining the written consent.
7.4 HAS THE ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR INSTITUTION IN CASE OF 7.3?
Yes, ethical clearance letter is enclosed.
8. / LIST OF REFERENCES:
1. Spijkervet F K L, Van Saene H K F, Panders A K, Vermey A, Mehta D M. Scoring irradiation mucositis in head and neck cancer patients. Journal of Oral Pathology and Medicine. 1989; 18: p167- 171.
2. Sonis S T. Pathophysiology of oral mucositis: novel insights and opportunities. Journal of supportive oncology October 2007;5: p3-11.
3. Ananthnarayan and Paniker. Textbook of Microbiology, Orient Longman Private Limited, 7th edition 2005: p75.
4. Parulekar W, Mackenzie R, Bjarnason G, Jordan R C K. Scoring oral mucositis. Oral oncology 1998;34: p63-71.
5. Hameed M A, Waqas S. Physiological basis and clinical utility of erythrocyte sedimentation rate. Pak J Med Sci 2006; 22: p214-217.
6. Stockman M A, Spijkervet F K L, Wymenga A N M, Burlage F R, Timens W, Roodenburg J L N, de Vries E G E,. Quantification of oral mucositis due to radiotherapy by determining viability and maturation of epithelial cells. Journal of Oral Pathology and Medicine 2002; 31: p153- 157.
7. Mohammed F, Poon I, Zhang L, Brownman G, Sathya J. Acute phase response reactants as an objective measure of mucosal toxicity in head and neck cancer patients undergoing radical radiation therapy with or without concurrent chemotherapy. International journal of radiation oncology 2006; 66: p436.
8. Hueber A J et al. Palifermin as treatment in dose-intense conventional polychemotherapy induced mucositis. Haematologica 2006; 91(7): p90-91.
9. Fassas A B, Micelli M H, Grazzlutti M, Dong L, Barlogie B, Anaissie E. Serial measurement of serum C-reactive protein levels can identify patients at risk for severe complications following autologous stem cell transplantation. Leukemia and Lymphoma Aug 2005;46: p1159-1161.
10. Yusuf T, Kenan I, Ismail A. Assessment of acute phase respose in acute ischemic stroke. Tohoku J Exp Med 2005; 206: p91 – 98.
11. Morlese J F, Forrester T, Jahoor F. Acute phase protein response to infection in severe malnutrition. American journal of physiology- endocrinology and metabolism 1998;275:p112- 117.
12. Ananian P, Hardwigsen J, Bernard D. Serum acute phase protein level as indicator for liver failure after liver resection. Hepatogastroenterology, 2005 May – June: 52(63): p857-61.
13. Boucher N E, Hanrahan J J, Kihara F Y. Occurrence of C- reactive protein in oral disease. Journal of dental research 1967 may-june:46(3): p624.
14. Sonis S T, Mucositis as a biological process: a new hypothesis for the development of chemotherapy – induced stomatotoxicity. Oral oncology 1998; 34: p39-43.
15. Kostler W J, Hejna M, Wenzel C, Zielinski C. Oral mucositis complicating chemotherapy and/or radiotherapy: Options for prevention and treatment. C A Cancer J Clin 2001;51: p 290.
9. / SIGNATURE OF THE CANDIDATE
10. /
REMARKS OF THE GUIDE
/ A feasible study11. / NAME & DESIGNATION OF (In block letters)
11.1 GUIDE / DR. PRATIMA S RAO
DEPARTMENT OF ORAL PATHOLOGY & MICROBIOLOGY,