RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
BANGALORE, KARNATAKA.
ANNEXURE II
SYNOPSIS FOR REGISTRATION OF SUBJECT FOR
DESSERTATION
1 / NAME OF THE CANDIDATEADDRESS FOR CORRESPONDANCE / Dr. SEETU PALO
Dr SEETU PALO,
POST GRADUATE STUDENT,
DEPARTMENT OF PATHOLOGY, BANGALORE MEDICAL COLLEGE & RESEARCH INSTITUTE, BANGALORE-560002
2 / NAME OF THE INSTITUTION / BANGALORE MEDICAL COLLEGE AND RESEARCH INSTITUTE, BANGALORE.
3 / COURSE OF STUDY & SUBJECT / M.D. IN PATHOLOGY
4 / DATE OF ADMISSION TO COURSE / 15 MAY 2012
5 / TITLE OF THE TOPIC / “UTILITY OF IMMUNOHISTOCHEMISTRY (HBME-1,CK19,S100) AND ALCIAN BLUE STAINING(PH 2.5) IN DISTINGUISHING HYPERPLASTIC, BENIGN AND PRIMARY MALIGNANT THYROID LESIONS-AN ANALYTICAL STUDY”
6) BRIEF RESUME OF THE INTENDED WORK:
6.1) NEED FOR STUDY
The management of various types of thyroid nodules depends on the accurate
diagnosis. Diagnostic dilemma is encountered even in histopathlogy such as papillary hyperplasia versus papillary carcinoma, hyperplastic versus neoplastic nodule (i,e follicular adenoma, follicular variant of papillary carcinoma, follicular carcinoma) and follicular adenoma versus minimally invasive follicular carcinoma. So, there is need to establish panel of ancilliary immunohistochemical markers that would aid in the diagnosis.
Recent literature(1,2,3,4) suggests that HBME-1 positivity,a mesothelial marker,indicates malignancy, where as diffuse CK19 staining confirms papillary differentiation. Papillary thyroid carcinoma(PTC) also shows S100 protein positivity (5,6 ) and presence of high density of S100 positive histiocytes indicates better prognosis in PTC(7). The proposed study will be establishing the diagnostic ability of the three selected immunohistochemical marker (i,e HBME-1, CK19, S100 ) in various thyroid lesions.
It is known that the luminal surface of tumor cells is covered by a glycoproteinaceous material that reacts positively with mucin stains(8). Our study also intends to investigate the efficacy of Alcian blue(pH 2.5) staining as a cheap diagnostic tool in difficult cases.
6.2) REVIEW OF LITERATURE
Recent studies have focused on identifying immunohistochemical markers that can help in differentiating benign from malignant lesions and follicular variant of papillary carcinoma (FVPTC) from follicular carcinoma(FC) or follicular adenoma(FA) or hyperplastic foci.
Cheung et al,(1) reported diffuse and moderate to strong CK19 staining in 80%(43/54) of PTCs and 57%(48/84) of FVPTC. 17%(6/35) of FA showed focal staining while none(0/4) of the FC were stained with CK19. 55% of PTC, 50% of FC, 67% of insular carcinoma and 50% of anaplastic carcinoma were HBME-1 positive where as all cases of nodular hyperplasia and FA were HBME-1 negative. Hence, they concluded that HBME-1 and CK19 positivity indicates malignancy and papillary differentiation respectively.
Debdas Bose et al,(2) suggested that focal CK19 may be found in benign disease but diffuse and strong positivity is characteristic of PTC and a negative CK19 staining is against the diagnosis of PTC.
Mustafa kosem et al,(5) found that PTC were stained 90% moderately and strongly with HBME-1; 88.3% moderately and strongly with CK19 and 48.4% moderately and strongly with S100.Out of 12 cases of follicular adenoma(FA),four cases revealed weakly positive CK19 staining; S100 was weakly positive in one.Among 5 cases of follicular carcinoma(FC),two were weakly CK19 positive and one moderately S100 positive.None of the cases of follicular adenoma and follicular carcinoma showed HBME-1 positivity.They concluded that HBME-1 is a highly specific and sensitive marker of PTC.
Studies by MR Nasr et al,(3) and Hussain A Saleh et al,(9) have also depicted the usefulness of HBME-1 & CK19 for the diagnosis of PTC.
Pattern of S100 protien immunostaining was studied by Kilicassian B et al,(6). 9/14 cases of PTC showed diffuse, 3/14 cases showed focal and 2/14 showed negative staining.All the 13 cases of papillary hyperplasia studied were negative for S100.
Damiani S et al,(10) proposed Alcian blue pH 2.5(AB 2.5) stain as an efficient tool in differentiating benign(which stained negatively with AB 2.5) from malignant papillae (15/17 stained positively with AB 2.5) in thyroid lesions.
6.3) OBJECTIVES OF THE STUDY
1. To evaluate the sensitivity and specificity of three immunohistochemical markers(HBME-1, CK19, S100) individually and in combination to differentiate between hyperplastic,benign and primary malignant thyroid pathology.
2. To study the diagnostic ability of Alcian blue( ph 2.5) staining in distinguishing benign and malignant thyroid lesion.
7) MATERIALS AND METHODS
7.1) SOURCE OF DATA
This study will be conducted on total and subtotal thyroidectomy and lobectomy specimens received in the Department of Pathology in Victoria hospital and Bowring and Lady Curzon Hospital, Bangalore during the period from November 2012 to October 2014.
7.2) METHODS OF COLLECTION OF DATA
A) STUDY DESIGN : Cross-sectional and analytical study.
B) PERIOD OF STUDY : November 2012 to October 2014.
C) PLACE OF STUDY : Department of Pathology, Victoria hospital and Bowring and Lady Curzon Hospital, Bangalore.
D) SAMPLE SIZE : minimum of 50 cases
E) INCLUSION CRITERIA:
Total and subtotal thyroidectomy and lobectomy specimens that will be histologically diagnosed as hyperplastic , primary benign and malignant neoplasms.
F) EXCLUSION CRITERIA:
1. Frank cases of colloid goitre, multinodular goitre , Grave’s disease and Hashimoto’s thyroiditis with no hyperplastic/malignant foci.
2. Cases of metastatic deposits in thyroid.
G) METHODOLOGY:
After obtaining approval and clearance from the institution’s ethical committee, those cases meeting inclusion and exclusion criteria will be included in this study. After macroscopic examination of the specimen and grossing, representative tissue bits will be fixed in 10% formalin, processed conventionally and embedded in paraffin blocks. Sections from these blocks will be stained with Haematoxylin & Eosin and Alcian blue stain(ph 2.5) according to standard procedures.Five micron sections from these blocks will also be immunostained using primary antibodies for HBME-1 (Dako, clone:M3505), CK19 (Biogenex, clone:RCK108), S100(Lieca, clone:S1/61/69) using the standard streptavidin biotin peroxidise technique.
H) ASSESSMENT TOOLS:
A systematic analysis of histomorphology on Haematoxylin & Eosin stained slides and positivity for Alcian blue (pH 2.5) will be done . A semiquantitative assessment of immunohistochemical scoring will be performed taking into account both the intensity and percentage of positive cells. The percentage of cells staining positively with HBME-1, CK19 and S100 will be scored as follows:
1) none of the cells stained - ‘0’
1) 1% -5% of the cells stained - ‘1’
2) 5% -25% of the cells stained - ‘2’
3) 25%-75% of the cells stained - ‘3’
4) 75%-100% of the cells stained - ‘4’.
The intensity will be scored as :
1) faint-‘0’ ,
2) intermediate – ‘1’ ,
3) intense-‘2’.
Positive controls will be pluera for HBME-1, skin for CK19, melanoma for S100 and appendix for Alcian blue(ph 2.5).The results will be recorded in the study proforma and statistically analysed.
I) STATISTICAL METHODS:
Data will be analysed using ‘Epi-info’ software and statistical tests like ‘Chisquare’ and ‘Anova’ will be used for analysis.
7.3) Does the study require any investigation or interventions to be conducted on patients or other human or animals? If so describe briefly
No
7.4) Has the ethical clearance been obtained from your institution?
Yes.
8) LIST OF REFERENCES
1) Carol C Cheung, Shereen Ezzat, Jeremy L Freeman, Irving B Rosen ,Sylvia L Asa. Immunohistochemical diagnosis of papillary thyroid carcinoma.Mod Pathol.2001:14: 338-42
2) Debdas Bose , Ram Narayan Das, Uttara Chatterjee, Uma Banerjee. CK19 immunoreactivity in the diagnosis of papillary thyroid carcinoma. Indian J Med Paediatr Oncol. 2012 Apr-Jun:32(2):107-11
3) M.R Nasr ,Sanjay Mukhopadhay , Shengle Zhange, Anna Luise A, Katzenstein . Absence of BRAF mutation in HBME-1+ & CK19+ atypical cell clusters in Hashimoto thyroiditis. Am J Clin Pathol. 2009:132: 906-912
4) Sunati Sahoo, Syed A Hoda, Juan Rosai, Ronald A DeLellis. Cytokeratin 19 immunoreactivity in the diagnosis of papillary thyroid thyroid carcinoma. Am J Clin Pathol. 2001:116: 696-702
5) Mustafa Kosem, Sabriye Polat, Mustafa Ozturk, Cetin Kotan, Hanefi Ozbek, Ekrem Algun.Differential diagnosis of papillary thyroid carcinoma:Immunohistochemical study of 112 cases. Eastern Journal Of Medicine :2005:10:15-19
6) Kilicarsian B, Pesterlli EH, Oren N, Sargin FC, Karpuzoglu G. EMA & S100 protein expression in benign and malignant papillary thyroid neoplasm. Adv Clin Path.2000 oct: 4(4) : 155-8
7) J.K.C Chan. Tumors of the thyroid and parathyroid glands in Christopher DM Fletcher’s Dignostic histopathology of tumors. Vol-2 . second edition.Churchill Livingstone.2000:959-1023
8) Juan Rosai, Giovanni Tallini. Thyroid gland in Rosai and Ackerman’s surgical pathology. vol-1. 10th edition.Elseiver Mosby.2011: 487-539.
9) Hussain A Saleh, Bo Jin , John Barnwell, Opada Alzohaili. Utility of immunohistochemical marker in differentiating benign from malignant follicular-derived thyroid nodules. Diagnostic Pathology. 2010 :5:9
10) Damiani S, Fratamico F, Lepertosa G, Dina R, Eusebi V. Alcian blue and epithelial membrane antigen are useful markers in differentiating benign from malignant papillae in thyroid lesions. Virchows Arch A Pathol Anat Histopathol. 1991:419(2): 131-5
9) / SIGNATURE OF THE CANDIDATE: /(DR.SEETU PALO)
10) / REMARKS OF THE GUIDE: / Histopathology is no doubt a powerful tool in diagnosing thyroid lesions.However , there are several overlapping features/pitfalls in accurate diagnosis of some of the lesions.So I do feel it is necessary at times to rely on ancilliary techniques like immunohistochemistry and special stains.Since no IHC marker is a gold standard in thyroid lesions,this study will be of great importance.Hence this study is recommended and forwarded.
11) / NAME AND DESIGNATION OF
11.1) / GUIDE: / DR. DAYANANDA. S. BILIGI, MD Professor, Department of Pathology
Bangalore Medical College & Research Institute, Bangalore.
11.2) / SIGNATURE:
11.3) / CO-GUIDE:
11.4) / SIGNATURE:
11.5) / HEAD OF THE DEPARTMENT: / DR. A. R. RAGHUPATHI, M.D Professor and H.O.D, Department of Pathology, Bangalore Medical College & Research Institute, Bangalore.
11.6) / SIGNATURE:
12) 12.1) REMARKS OF THE CHAIRMAN AND PRINCIPAL:
12.2) SIGNATURE: