Bangalore,karnataka proforma for registration of subjects of dissertation



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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE ,KARNATAKA

PROFORMA FOR REGISTRATION OF SUBJECTS OF DISSERTATION


1.

Name of the candidate

&address


Dr .Suba .G ,

Dr.B.R. Ambedkar Medical College,

K.G. Halli,

Bangalore-560045




2.

Name of the institution

Dr .B .R .Ambedkar medical college


3.

Course of study & subject

M. D. Pathology

4.

Date of admission to course

31-05-2010

5.

Title of the topic

“HISTOMORPHOLOGICAL STUDY OF PROSTATIC TURP (trans urethral resection of prostate) SPECIMENS WITH SPECIAL REFERENCE TO PROSTATIC INTRAEPITHELIAL NEOPLASIA BY USING IMMUNOHISTOCHEMISTRY.”


6.

Brief resume of the intended work

6.1 Need of the study Annexure I

6.2 Review of Literature Annexure II

6.3 Objectives of the study Annexure III



7.

Materials,Method

and Design



7.1 Source of Data Annexure IV

7.2 Method of collection Annexure V

7.3 Study design Annexure VI

7.4 Has Ethical clearance been obtained

from your institution? Annexure VII


8.

List of References


Annexure VIII


9.

Signature of the candidate




10.

Remarks of the Guide

Prostatic intraepithelial neoplasia requires confirmation by doing Immunohistochemistry after making a diagnosis by hematoxylin and eosin sections. This helps in accurate diagnosis , early treatment and better prognosis for the patient.


11.1

Name & Designation of the guide

Dr. B .N. Swarnagowri,

Associate Professor,

Department of Pathology


11.2

Signature




11.3


Name & Designation of the co-guide

Dr.M.G.Desai,

Associate professor and HOD,

Department of urology, Dr. B. R. Ambedkar medical college.


11.4

Signature





11.5

Head of the department

Dr.Y.A .Manjunatha,

Professor and Head,

Department of Pathology.


11.6

Signature




12.1

Remarks by the Chairman & Principal




12.2

Signature




Annexure I

BRIEF RESUME OF THE INTENDED WORK.

6.1 NEED OF THE STUDY:

Prostate cancer is a leading cause of morbidity and mortality worldwide(1)

The incidence of prostate carcinoma is increasing with age. It increases from 20% in men in

their 50s, to approximately 70% in men between the ages 70 and 80 yrs.

In view of the increased life expectancy the absolute number of clinically significant prostate

carcinomas will probably increase in the coming decades . To prevent an equal rise in

mortality, early detection of prostate cancer will become essential , as will the detection of

premalignant lesions such as prostatic intraepithelial neoplasia.(2)

Prostatic intraepithelial neoplasia is now considered to be the most likely precursor of

prostate cancer. Prostatic Intraepithelial Neoplasia lesions can only be diagnosed by

histopathological examination of prostatic tissue . It is impossible to detect Prostatic

Intraepithelial Neoplasia clinically by direct rectal examination, prostate specific antigen

assay or ultrasound.(2)

Different biochemical markers can be used to identify Prostatic Intraepithelial

Neoplasia. These markers can be used to develop diagnostic and therapeutic strategies.

This study is undertaken to know the percentage of Prostatic Intraepithelial Neoplasia in

TURP (trans urethral resection of prostate ) specimens in the locality in male patients of

more than 40yrs.

Annexure II

6.2 REVIEW OF LITERATURE:

INTRODUCTION:

The prostate is a retroperitoneal organ encircling the neck of the bladder and urethra.

Histologically the prostate is composed of glands lined by two layers of cells, a basal

layer of low cuboidal epithelium covered by a layer of columnar secretory cells.

.PROSTATIC INTRAEPITHELIAL NEOPLASIA:

Prostatic Intraepithelial Neoplasia is the currently preferred term for a process involving

prostatic ducts and acini, which has also been described as intraductal or ductal –acinar

dysplasia.(3).

Prostatic Intraepithelial Neoplasia consists of architecturally benign prostatic acini lined

by cytologically atypical cells with prominent nucleoli. It involves larger branching glands

with papillary infolding .Glands are surrounded by a patchy layer of basal cells and an

intact basement membrane.(4)

The earliest report on premalignant lesions date back to 1926 . No

distinction between prostatic intraepithelial neoplasia and lesions resembling prostatic

intraepithelial neoplasia was made at that time.(2) In 1965, Mc Neal described different

lesions with possible premalignant features in prostatic epithelium. But it would last

until 1986 when Mc Neal and Bostwick described the first reproducible criteria for the

diagnosis of intra ductal neoplasia. A classification into 3 grades was proposed.(2)

One year later , Bostwick and Brawer proposed to change the terminology

to prostatic intraepithelial neoplasia. Finally , in 1989 , at a workshop on premalignant

lesions of the prostate , the classification was changed into low grade [ former grade 1 ]

and high grade [ former grades 2 and 3 ].(2)

CARCINOMA PROSTATE :

Carcinoma prostate is typically a disease of men over 50 yrs.

Histologically prostate cancer glands are more crowded , and characteristically lack

branching and papillary infolding. The outer basal cell layer typical of benign glands is

absent. The cytoplasm of the tumour cells ranges from pale-clear as seen in benign glands to

a distinctive amphophilic appearance . Nuclei are large and often contain one or more large

nucleoli . (4)

PIN AS A PRECURSOR LESION OF PROSTATE CANCER :

PIN and prostate carcinomas are mostly multifocal and preferentially found in the

peripheral zone of the prostate. In an autopsy study of Haggman , high grade PIN

was found in 63% solely in peripheral zone , in 36% in the peripheral and transitional

zone and in 1% solely in the transition zone.(5 ) These findings are similar to the zonal

distribution of prostate carcinoma which originates from the peripheral zone in 75 – 80%

and from the transition zone in 25 – 25% .

A growing body of data demonstrates that the expression of various biomarkers in high grade

PIN is either 1. The same in highgrade PIN and carcinoma prostate unlike in benign

prostatic tissue or 2. intermediate between benign tissue and carcinoma. All these findings

are expected if high grade PIN is a precursor lesion to carcinoma prostate .(6)

The finding of high grade PIN on TURP also appears to place men at higher risk of

harbouring cancer .(7)

DETECTION OF PIN LESIONS BY USING IMMUNOHISTOCHEMISTRY:

ImmunoHistoChemistry techniques have been used since the 1940s , when AH Coons

and colleagues published their landmark paper describing the identification of tissue

antigens using a direct fluorescence protocol.(8)

Principle of the procedure:

Antigen detection in tissues and cells is a multistep immunohistochemical process. The initial

Step binds the primary antibody to its specific epitope. After labelling the antigen with a

primary antibody , a secondary antibody is added to bind to the primary antibody. An enzyme

label is then added to bind to the secondary antibody; this detection of the bound antibody is

evidenced by a colorimetric reaction.

P504S (α –methylacyl – coA racemase ) and p63 markers:

α-methylacyl – coA racemase is a mitochondrian and peroxisomal enzyme

involved in the metabolism of branched – chain fatty acid and bile acid intermediates. (9)

Several investigators have demonstrated that immunostaining for P504S, a

cytoplasmic protein that is overexpressed in a high percentage of prostate cancers and in

many cases of high grade prostatic intraepithelial neoplasia , can also be of use in the

diagnosis of prostate biopsies . P504S can be used as a positive marker for PIN .(10)

p63 :


The p53 homologue p63 encodes for different isotypes able to either transactivate p53

reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63).  Prostate basal cells

in culture predominantly express the DeltaNp63alpha isotype . In contrast, p63 protein is

not detected in human prostate adenocarcinomas.(11)  p63 may be used to stain tissue sections

for the presence of basal cells, recognizing that PIN retains an intact or fragmented basal

cell layer whereas cancer does not.(12)

Immunohistochemical staining for P504S and p63 are performed on all

specimens which are fixed in 10% formalin solution , and routinely processed for

embedding in paraffin . Tissue sections were deparaffinised in xylene and rehydrated through

ethanol.

To overcome cross-linking due to formalin fixation , the tissue sections were placed in

an epitope retrievel solution and heat treated in a water bath followed by a cool –down

period at room temperature.The tissue sections are immunostained for P504S and p63 using

an automated staining system. In brief, all sections are made reacted to hydrogen peroxide

solution to block endogenous peroxidase activity and then thoroughly rinsed in a washing

buffer.


The sections are incubated with anti-P504S and anti p63 antibodies and then washed with

the washing buffer. Bound antibodies were detected by incubation with dextran polymer

reagent conjugated with peroxidase and secondary antibody. The tissues are then washed

with the washing buffer .Color development was achieved with 3 ,3’-diaminobenzidine.

The tissues are counterstained with hematoxylin.

Annexure III



6.3 OBJECTIVES OF THE STUDY :

To evaluate the percentage of prostatic intraepithelial neoplasia in TURP (trans urethral

resection of prostate ) specimens and confirm it by using immunohistochemistry.

To determine the therapeutic benefits.



7. MATERIALS , METHODS AND DESIGN :

Annexure IV



7.1 SOURCE OF DATA:

All cases of transurethral resection of prostate forms the material source.

This is a prospective study being carried out in the department of pathology at

DR . B . R . AMBEDKAR MEDICAL COLLEGE , BANGALORE.

INCLUSION CRITERIA : Male patients of more than 40yrs.

Patients with family history of prostate carcinoma.

EXCLUSION CRITERIA : Cases of metastatic tumour deposits in the prostate.

SAMPLE VOLUME : Minimum of fifty cases collected from December 2010

to August 2012 are included.

Annexure V :



7.2 METHOD OF COLLECTION:

The specimens are received in 10% formalin . Following scrutinizing the patient details and

identity , the specimen of TURP chips are fixed in fresh formalin for 24hrs. Gross

description of all the fragments and process all the fragments for microscopic study

by using hematoxyline and eosin staining and confirmed by doing immunohistochemistry.

HEMATOXYLIN & EOSIN STAINING PROCEDURE :

1.Dewax sections, hydrate through graded alcohol in water.

2.Remove formalin pigment.

3.stain in alum hematoxyline.

4.wash in running tap water until sections blue for five minutes or less.

5.Differentiate in 1% acid alcohol for 5-10 seconds.

6. wash well in tap water until sections are again blue.

7.Blue by dipping in an alkaline solution(ammonia water) followed by a tap water wash.

8.counterstained in 1% Eosin Y for 10 minutes. Wash in running tap water for 1-5 mts.

9.Dehydrate , clear and mount in DPX.

Result: nucleus :blue, cytoplasm and connective tissue shades of pink.

Interpretation of the slide:

Cytologically , low grade PIN and high grade PIN have clear and reproducible features.(2)

Low grade PIN High grade PIN

Architecture - Epithelial cell crowding - Similar to low grade PIN,

and stratification , with more crowding and stratification.

irregular spacing. Four patterns: tufting , micro

papillary, cribriform and flat.

Cytology

Nuclei - Enlarged , with marked - Enlarged , some size and shape

size variation . variation.

Chromatin - Normal - Increased density and clumping .

Nucleoli - Rarely prominent - occasionally to frequently large

and prominent similar to invasive

carcinoma ; sometimes multiple .

Basal cell layer - Intact - may show some disruption.

Basement

Membrane -Intact - Intact .



IMMUNOHISTOCHEMISTRY PROCEDURE:

Tissues are processed for paraffin embedding similar to routine processing of tissues

for microscopic study.

The tissue sections from paraffin block are cut with 1 micro thickness and float

in water heated to 40-450C .

The tissue sections are taken on to a PLL coated slide with a positive control tissue

section.

The tissue sections are placed on a slide warmer for deparaffinisation and further

deparaffinisation using xylene .

Other sequential steps are depicted in the flow chart.

The antigen retrieval uses micro oven processing as a standard protocol.

The antibodies and other consumables are obtained from Biocare.

The stained tissue sections with the positive control are ready for interpretation

and final reporting using the regular Binocular microscope.

Interpretation of slide:

Cellular localization : granular cytoplasm (P504S) and nuclear (p63).

p63 is detected in prostatic basal cells in normal prostate . Prostatic Intraepithelial

Neoplasia retains an intact or fragmented basal cell layer whereas cancer does not.

P504S staining is present in premalignant and malignant lesions of prostate but not in benign

prostatic tissue.

P504S immunoreactivity was measured negative (0) , weakly positive ( +1),

moderately positive (+2) , and strongly positive (+3) .(9)

Annexure VI

7.3 STUDY DESIGN :

PROSPECTIVE STUDY

Annexure VII

7.4 . Has ethical clearance been obtained from the institution ?.

Annexure VIII

LIST OF REFERENCES :

1.Routh JC, Leibovich BC Adenocarcinoma of the prostate :epidemiological trends,

Screening,diagnosis and surgical management of localized disease. Mayo clin proc. 2006

Jan;81(1):132.

2. S. Joniau, L.Goeman,J.Pennings,H.Van poppel. Prostatic intraepithelial neoplasia

Importance and clinical management accep 10mar2005,publ online 24 mar 2005

379-385.

3.Brawer MK. Prostatic intraepithelial neoplasia:A premalignant lesion. Hum Pathol 1992,

23:242-248.

4.Robbins and Cotran Pathologic Basis of Disease, eighth edition ,pg 998-999.


5. M.J. Haggman, J.A. Macoska, K.J. Wojno, J.E. Oesterling. The relationship between
prostatic intraepithelial neoplasia and prostate cancer: critical issues. J Urol 158 (1997)
(12 - 22)
6.Walsh Retik Vaughan Wein Campbell’s urology Eighth edi . vol.4 , pg 3025.

7.Pacelli A,Bostwick DG ;clinical significance of high grade prostatic intraepithelial


Neoplasia in transurethral resection specimens .Urology 1997;50:355-359.

8.Coons AH, Creech HJ , Jones RN(1941) Immunological properties of an antibody

containing a fluorescent group.Proc Soc Exp Biol Med 47:200-202.

9. Chin-Lee Wu, MD, PhDa, Ximing J Yang, MD, PhD bc , Maria Tretiakova, MD,


PhD b, Kurt T Patton, MD c, Elkan F Halpern, PhD e, Bruce A Woda, MD d, Robert
H Young, MD a,Zhong Jiang, MD d .Analysis of α-methylacyl-CoA racemase (P504S)
expression in high-grade prostatic intraepithelial neoplasia . Humpath Vol 35Issue 8,
Pages 1008-1013 (Aug 2004) .







10. Tacha DE, Miller RT Use of p63/P504S monoclonal antibody cocktail in



immunohistochemical staining of prostate tissue Appl immunohistochem



Mol Morphol.2004 mar;12(1):75-8.



11. Signoretti S, Waltregny D, Dilks J, Isaac B, Lin D, Garraway L, Yang A

 

Montironi R, McKeon F, Loda M .p63 is a prostate basal cell marker and is required for



prostate development. Am J Pathol. 2000 Dec;157(6):1769-75.





12. David G Bostwick  and Junqi Qian High-grade prostatic intraepithelial neoplasia



Modern Pathology (2004) 17, 360–379, adv online publication, 23 January 2004;

doi:10.1038/modpathol.3800053.















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