RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE ,KARNATAKA
PROFORMA FOR REGISTRATION OF SUBJECTS OF DISSERTATION
1. Name of the Dr .Suba .G , candidate Dr.B.R. Ambedkar Medical College, &address K.G. Halli, Bangalore-560045
2. Name of the Dr .B .R .Ambedkar medical college institution
3. Course of study & M. D. Pathology subject 4. Date of admission 31-05-2010 to course 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 6.1 Need of the study Annexure I intended work 6.2 Review of Literature Annexure II
6.3 Objectives of the study Annexure III
7. Materials,Method 7.1 Source of Data Annexure IV and Design 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 Prostatic intraepithelial neoplasia requires confirmation by Guide 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. Name & Dr. B .N. Swarnagowri, 1 Designation of the Associate Professor, guide Department of Pathology
11. Signature 2
11. Name & 3 Designation of the Dr.M.G.Desai, co-guide Associate professor and HOD, Department of urology, Dr. B. R. Ambedkar medical college.
11. Signature 4
11. Head of the Dr.Y.A .Manjunatha, 5 department Professor and Head, Department of Pathology.
11. Signature 6
12. Remarks by the 1 Chairman & Principal
12. Signature 2 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 b c , 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 35, Issue 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.