Rajiv Gandhi University of Health Sciences, Karnataka,

Bangalore.

“ESTROGEN, PROGESTERONE AND HER-2/neu STATUS IN BREAST CARCINOMA: COMPARISON OF IMMUNOCYTOCHEMISTRY AND IMMUNOHISTOCHEMISTRY.”

Name of the candidate :Dr. SUBHAN ALI R.

Guide :Dr. HILDA FERNANDES

Course and Subject :M.D (Pathology)

Department of Pathology,

Father Muller Medical College,

Kankanady, Mangalore – 575002.

AUGUST – 2012

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA

PROFORMA FOR REGISTRATION OF THE SUBJECT FOR

DISSERTATION

1. / NAME OF THE CANDIDATE AND ADDRESS / DR.SUBHAN ALI R.
P.G. RESIDENT,
FR. MULLER MEDICAL COLLEGE, KANKANADY,
MANGALORE – 575002.
2. / NAME OF THE INSTITUTION / FR. MULLER MEDICAL COLLEGE, KANKANADY,
MANGALORE – 575002
3. / COURSE OF STUDY AND SUBJECT / M.D. (PATHOLOGY)
4. / DATE OF ADMISSION TO COURSE / 15-05-2012
5. / TITLE OF THE TOPIC:
Estrogen, Progesterone and HER-2/neu status in breast carcinoma: Comparison of Immunocytochemistry and Immunohistochemistry.

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6. / Brief resume of the intended work:
6.1 Need for the study
Breast cancer is the second most common cancer among Indian females, next only to cervical cancer. However breast cancer cases are found to be spiraling the world over and urban India is no exception. On the other hand, cases of cervical cancer, which is the most common form of cancer among Indian women is found to be dipping in some cities by almost 50%. It has been predicted that by 2020, breast cancer will overtake cervical cancer as the most common type of cancer among women in India.
In the past few decades, the importance of fine needle aspiration cytology (FNAC) has been well documented in the diagnosis of breast lesions. Fine needle aspiration has become a standard technique in evaluation and frequently, the source of primary diagnosis.(1)
The introduction of immunocytochemistry (ICC) opened the gateway to a new era of pathology, in which immunolocalization of specific cellular antigens has moved from being a purely scientific research tool to becoming an invaluable diagnostic adjunct in most pathology laboratories. Some tumors, notably carcinoma of breast and prostate, are often responsive to hormones, a property that has been exploited by the use of endocrine surgery and medically, using drug therapy to influence hormone levels on or inhibit the effects of hormones on tumor cells. Trucut biopsy and immunohistochemistry(IHC) is currently used to determine the locally advanced lesions for preoperative chemotherapy. Formalin may destroy some epitopes in paraffin-embedded tissues and hence ICC can be a better alternative as it results in more adequate fixation of cells.(2)
The ICC could be useful for inoperable tumors or in patients at high risk for surgery and in patients with advanced tumors. It is also used as an indication for radiotherapy or chemotherapy prior to surgical treatment, when a determination of hormonal receptors is desired and to establish the probable mammary origin of metastatic disease.(3)

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6.2 Review of Literature
The IHC or ICC, is a method for localizing specific antigens in tissues or cell based on antigen-antibody recognition; it seeks to exploit the specificity provided by the binding of an antibody with its antigen at a light microscopic level. The history of IHC dates back to 1941 when Coons and colleagues labeled an antibody with fluorescent dye and used it to identify an antigen in tissue sections. Since the 1970s the use of IHC techniques has taken off exponentially, in parallel with the development of specific molecular markers. In Oxford, Taylor and Burns developed the first successful demonstration of antigens in routinely processed formalin-fixed paraffin-embedded tissues.(4)
ICC has over the years evolved into a revolutionary diagnostic tool for the pathologist. This prompted the development of special staining techniques to stain cells of particular lineage; marking the beginning of histochemistry in the mid nineteenth century. Advent of aniline dyes revolutionized immunochemistry from 1862 to 1929. The ICC data have to be considered together with the other information available to the cytopathologist. ICC should never be selected as the sole or only method of diagnosis but must be integrated into the cytologic decision making process.(5) ICC is used for analysis of tumour origin and to predict the behavior of the tumour by identifying the prognostic markers.
The ICC determination of estrogen receptor(ER) and progesterone receptor(PR) in routinely fixed material is simple and reliable, especially for ER with sensitivity 96.1% and specificity 86.9% and global cytohistologic correlation of about 94% as concluded by Zoppi et al in 2002(3). In a study conducted by Sasaki et al correlation between Dextran-coated charcoal(DCC) and ICC was 81% for ER and 74% for PR, that between enzyme immunoassay and ICC was 88% for ER and 80% for PR.(6)
ER and PR positivity rates as well as staining scores compared between the scarpe smears and tissue sections by Malaviya et al, showed concordance between cytology and histology of 84% for ER and 90% for PR. Both positivity rates and staining intensity scores were higher for cyochemistry than for histochemistry.(7)
In another study the concordance for ER was 50% and for PR was 29% and the use of least best buffer and technical errors contributed to the lower ICC rates. An overall accuracy of 91.1% for estrogen receptor and 88.9% for progesterone receptor was observed in a study conducted by Nasreen et al.(8)
Beatty et al studied HER-2 receptor status by ICC, IHC and FISH in FNAC specimens and formalin-fixed,paraffin-embedded tissue samples obtained from surgically resected breast cancers respectively.(9)
The Concordance between cytology and histology was 98% for ER, 95% for PR, and 100% for HER2/neu in a study conducted by Moriki et al.(10)
The crucial question that remains unanswered in the literature is if a case is immunocytochemistry-positive and immunohistochemistry-negative, should the patient receive benefit of hormonal therapy.
6.3 Objective of the study:
To evaluate the degree of correlation between immunocytochemistry (ICC) and immunohistochemistry (IHC) of ER, PR and HER-2/neu in Breast cancer.
7. / Materials and Methods
7.1 Source of data:
The patients with primary breast carcinoma undergoing surgery at Father Muller Medical College Hospital will be included in this study.
·  Sample size: A minimum of 30 cases.
·  Duration of the study: The study period will be two years.
7.2  Method of collection of data:
The patients with primary carcinoma of breast undergoing surgery will be included in the study for a period of 2 years from June 2012 to May 2014.
The clinical details like age, size of the lump will be taken. Clinicians will be instructed to submit the specimens to the laboratory immediately following the surgery. The tumor will be identified and FNAC will be performed using a 23-gauge needle and 10 cc disposable syringe. In cases where lump is not located, serial sections of mastectomy specimens will be performed to identify the lump. Scrape smears will be taken with the help of a clean scalpel blade. Smears will be fixed immediately in cold acetone for about 10 mins and stored in the refrigerator for ICC. Paraffin embedded sections will be taken for histopathology and IHC. For ICC and IHC, the technique adopted is according to manufacturer’s instructions (BioGenex). Monoclonal antibodies will be used from manufacture BioGenex. Anti- Estrogen Receptor, Anti- Progesterone Receptor and Anti-ErbB-2/ HER-2. The principle adopted is super sensitiveTM IHC detection system.
For IHC method, the FFPE sections will be dewaxed in xylene and passed through graded alcohols; sections will be treated with methanol and hydrogen peroxide. The remaining steps followed same as ICC. Appropriate negative and positive controls will be run with every batch of tests samples. The smears with >100 cells will be considered adequate. Smears in which at least 5% of tumor nuclei stained brown to yellow will be considered positive. Sections will be interpreted as positive if at least 10% of the tumor nuclei stained positive. A semiquantitative method described by Shousha et al, based on intensity of nuclear staining and distribution of positive nuclei will be used to score the smears and sections.(11)
SELECTION CRITERIA
a. Inclusion Criteria
The patients with primary carcinoma of breast undergoing surgery in Father Muller Medical college.
b. Exclusion Criteria
·  The patients whose specimens are fixed in formalin.
·  Necrotic and highly desmoplastic tumors with scanty cellularity.
·  Extensive in situ comedo component.
·  Mucinous carcinomas.
Type of study
Comparative study
Statistical Analysis
1. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy will be calculated.
2. The correlation between ICC and IHC analyzed by using kappa statistics.
7.3 Does the study require any investigations or interventions to be conducted on patients or other humans or animals?
Yes
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
Yes
8. / REFERENCES:
1.  Bhargava V, Jain M, Agarwal K,Thomas S, Singh S. Cytological nuclear grading in carcinoma breast.J cytol. 2008;25(2):58-61.
2.  Radhika K, Prayaga AK. ER PR receptor status in breast carcinoma. Ind J Cancer. 2010;47(2):148-50.
3.  Zoppi JA, Rotundo AV, Sundblad AS. Correlation of immunocytochemical and immunohistochemical determination of estrogen and progesterone receptors in Breast cancer. Acta cytol. 2002;46(2):337-40.
4.  Taylor CR, Shi SR, Barr N .Techniques of immunohistochemistry: Principles, Pitfalls, and Standardization. In: Dabbs DJ .Diagnostic Immunohistochemistry. Third edition. Philadelphia. Saunders;2010;1-41.
5.  Coleman R. The impact of histochemistry-a historical perspective. Acta Histochem. 2000;102(1):5-14.
6.  Sasaki M, Morimoto K, Koh M, Wakasa K, Haba T, Kinoshita H. ERs and PRs in breast Imprints. Acta cytol. 2002;46(6):1056-60.
7.  Malaviya A, Chinoy R, Prabhudesai NM, Sawant MH, Parmar V, Badwe RA. Immunocytochemistry on Scrape cytology in breast cancer. Acta cytol.2006;50(3): 284-90.
8.  Hafez NH, Tahoun NS. Assessment of the Reliability of Immunocytochemical Detection of Estrogen and Progesterone Receptors Status on the Cytological Aspirates of Breast Carcinoma. Journal of the Egyptian Nat. Cancer Inst.2010; 22( 4): 217-25.
9.  Beatty B G, Bryant R, Wang W, Wang W, Ashikaga T, Gibson PC, Leiman G, Weaver DL.HER-2 Detection by FISH and Immunocytochemical Analysis on Breast FNA Specimens. Am J Clin Pathol.2004;122:246-55.
10.  Moriki T, Takahashi T, Ueta S, Mitani M, Ichien M. Hormone receptor status and HER2/neu over expression determined by automated immunostainer on routinely fixed cytologic specimens from breast carcinoma: correlation with histologic sections determinations and diagnostic pitfalls. Diagn Cytopathol. 2004;30(4):251-6.
11.  Shousha S, Coady AT, Stamp T, James KR. Oestrogen receptors in mucinous carcinoma of breast:An immunohistologic study using paraffin wax sections. J Clin Pathol 1989;42:902-05.
9. / SIGNATURE OF THE CANDIDATE:
10. / REMARK OF THE GUIDE:
11. / NAME AND DESIGNATION OF (in block letters)
11.1 GUIDE / DR.HILDA FERNANDES MD.
PROFESSOR,
DEPARTMENT OF PATHOLOGY,
FATHER MULLER MEDICAL COLLEGE, KANKANADY,
MANGALORE-575002.
11.2 SIGNATURE
11.3 CO-GUIDE / DR.RAPHAEL HART LYNGDOH MD.
ASSISTANT PROFESSOR,
DEPARTMENT OF PATHOLOGY,
FATHER MULLER MEDICAL COLLEGE, KANKANADY,
MANGALORE-575002.
11.4 SIGNATURE
11.5 HEAD OF THE
DEPARTMENT / DR. MUKTHA R. PAI MD.
PROFESSOR AND HOD,
DEPARTMENT OF PATHOLOGY,
FATHER MULLER MEDICAL COLLEGE, KANKANADY,
MANGALORE-575002
11.6 SIGNATURE
12. / 12.1 REMARKS OF THE CHAIRMAN AND DEAN
12.2 SIGNATURE

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