RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
PROFORMA FOR REGISTRATION OF SUBJECTS OF DISSERTATION
1. / NAME OF CANDIDATE AND ADDRESS / DR.NITYA PRABHAKARANDR.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 / 31-5-2012
5. / TITLE OF THE TOPIC / “HISTOMORPHOLOGICAL EVALUATION OF GASTROINTESTINAL MALIGNANCY DIAGNOSED BY ENDOSCOPY WITH SPECIAL REFERENCE TO EPIDERMAL GROWTH FACTOR RECEPTOR BY IMMUNOHISTOCHEMISTRY”
6. / BREIF RESUME OF THE INTENDED WORK / 6.1 NEED OF THE STUDY ANNEXURE I
6.2 REVIEW OF LITERATURE ANNEXURE II
6.3 AIMS AND OBJECTIVES ANNEXURE III
7. / MATERIALS AND METHODS / 7.1 SOURCE OF DATA ANNEXURE IV
7.2 METHOD OF COLLECTION ANNEXURE V
7.3 STAINING TECHNIQUES ANNEXURE VI
8. / LIST OF REFERENCES / ANNEXURE VII
9. / SIGNATURE OF THE CANDIDATE
10. / REMARKS OF GUIDE / THIS IS A STUDY OF EGFR OVER EXPRESSION BY (IHC) IN GASTROINTESTINAL MALIGNANCIES.IT WOULD DEFINITELY HELP IN BETTER UNDERSTANDING OF PROGRESSION OF DISEASE AND THUS PLANNING THE STRATEGY FOR PATIENT TREATMENT.
11.1 / NAME AND DESIGNATION OF THE GUIDE / DR. SHAISTA CHOUDHARY
ASSOCIATE PROFESSOR
DEPARTMENT OF PATHOLOGY
DR.B.R AMBEDKAR MEDICAL COLLEGE, BANGALORE -560045
11.2 / SIGNATURE
11.3 / HEAD OF DEPARTMENT / DR. MANJUNATHA Y.A
PROFESSOR AND HEAD OF DEPARTMENT
PATHOLOGY,
DR. B.R AMBEDKAR MEDICAL COLLEGE, BANGALORE-560045
11.4 / SIGNATURE
11.5 / NAME AND DESIGNATION OF CO GUIDE / DR. B.G. MANJUNATH, PROFESSOR, DEPARTMENT OF SURGERY, DR B.R.AMBEDKAR MEDICAL COLLEGE,BANGALORE -560045
11.6 / SIGNATURE
12.1 / REMARK AND SIGNATURE OF PRINCIPAL
6. BRIEF RESUME OF INTENDED WORK
ANNEXURE I
6.1 NEED FOR STUDY
Gastricadenocarcinoma is the most common malignancy of the stomach, comprising over 90% of all gastric cancers. Early symptoms resemble those of chronic gastritis, including dyspepsia, dysphagia, and nausea. As a result, these tumours are often discovered at advanced stages, when symptoms such as weight loss, anorexia, altered bowel habits, anaemia, and haemorrhage trigger further diagnostic evaluation.
Adenocarcinoma of the colon is the most common malignancy of the GI tract and is a major cause of morbidity and mortality worldwide. In contrast, the small intestine, which accounts for 75% of the overall length of the GI tract, is an uncommon site for benign and malignant tumours. Among malignant small intestinal tumours, adenocarcinomas and carcinoid tumours have roughly equal incidence, followed by lymphomas and sarcomas. The availability of endoscopic screening combined with the knowledge that most carcinomas arise within adenomas presents a unique opportunity for cancer prevention. Unfortunately, colorectal cancers develop insidiously and may therefore go undetected for long periods. Cecal and other right-sided colon cancers are most often called to clinical attention by the appearance of fatigue and weakness due to iron deficiency anaemia. Thus, it is a clinical maxim that the underlying cause of iron deficiency anaemia in an older man or postmenopausal woman is GI cancer until proven otherwise. Left-sided colorectal adenocarcinomas may produce occult bleeding, changes in bowel habits, or cramping left lower quadrant discomfort.
Although histology and exfoliative cytology remain the most commonly used methods in the diagnosis of cancer, new techniques are being constantly added to the tools of the surgical pathologist. Some, such as immunohistochemistry, are already well established and widely used; others, including molecular methods, are rapidly finding their way into the “routine” category.
ANNEXUREII
6.2 REVIEW OF LITERATURE.
Gastric cancer is the 5th most common cancer among males and 7th most common cancer among females in India1. The aggressiveness of the disease and need for improvement in therapeutic options is discerned by the fact the gastric cancer is the 2nd most common cause of cancer deaths globally2.
Bacterial and viral infections leading to chronic inflammation play a major role in gastrointestinal carcinogenesis3.
The pathogenesis of gastric cancer involves multiple risk factors including dietary , infections , occupational , genetic and preneoplastic risk factors , most of which act on the gastric mucosal environment over a prolonged period. The resultant sequential changes in the gastric mucosa that precede the development of invasive cancer are known as the ‘precancerous cascade’ first described in 19754, where normal gastric mucosa is transformed by chronic atrophic gastritis and develops multifocal atrophy and intestinal metaplasia, followed by the appearance of dysplasia and finally invasive carcinoma5.
The primary epithelial tumour of the stomach is the adenocarcinoma, and develops from the stomach mucosa, usually maintaining glandular differentiation5.
Endoscopic screening is considered to be the most sensitive and specific test for gastric cancer. Dysplasia may present as a flat lesion or exhibit polypoid growth, with depressed, reddish or discoloured mucosa. Endoscopic detection of changes in colour, relief and architecture of the mucosal surface enables the classification of gastric cancers according to their macroscopic growth pattern. Early gastric cancer may feature protruded (Type I ) , elevated (Type IIa) , flat (Type II b) , depressed (Type II c) or excavated (Type III) growth3 , whereas advanced gastric carcinomas are classified into polypoid (Type I) , fungating (Type II) , ulcerated ( type III ) or infiltrative (type IV) growth patterns3
Malignant primary gastric cells at first confined into the mucosal layer (intra-mucosal cancers) and after a rather long natural history progress infiltrating the other layers of the wall stepwise (submucosa, muscularis propria, subserosa and serosa). When at diagnosis the cancer invasion is confined to mucosa or submucosal layer is defined as early gastric cancer (EGC), whereas invasion into muscularis propria or beyond it is defined as advanced gastric cancer (AGC)6.
Various systems have been applied to the classification of gastric carcinoma, including the WHO3, Ming5 , Lauren7, and Goseki5 classifications. The clinical significance of these classifications is limited, with only the Lauren and perhaps the Goseki classifications providing prognostic assessments5 However the Lauren classification has been the most successful system, as it defines two distinct histological entities, which clearly exhibit different clinical and epidemiological characteristics, even in advanced gastric cancers5.
In the Lauren classification7 intestinal- type carcinomas maintain the glandular phenotype, with well – to moderately –differentiated tumours forming identifiable glands , often with poorly differentiated tumour cells at the invasive front Typically arising on a background of intestinal metaplasia , these tumours exhibit an intestinal , gastric and gastrointestinal mucinous phenotype. Diffuse- type carcinomas form no or very few glandular structures , instead usually infiltrating the gastric wall , appearing diffusely distributed as small , round single cells or poorly cohesive cell clusters. They may resemble signet-ring cells, and may contain small amounts of intestinal mucin5.
Colorectal cancer is one of the most common forms of gastrointestinal malignancies in the world8. Compared to the western world , the incidence rates of colorectal cancer are low in India ; for colon cancer they vary from 0.7 to 3.7 / 100,000 among men and 0.4 to 3 / 100,000 among women and for rectal cancer from 1.6 to 5.5/ 100,000 among men and 0.6 to 2.8 / 100,000 among women9.
Colorectal cancer in Indians occurs at a younger age and is often distal to the splenic flexure. Colorectal cancer should be suspected irrespective of the patient’s age, and as simple measures like sigmoidoscopy may pick up majority of cases of colorectal cancers in India 8.
Colon cancer arises from mucosal polyps. The 2 most common histological types are hyperplastic and adenomatous. Histologically, hyperplastic polyps contain an increased number of glandular calls with reduced cytoplasmic mucus, but lack nuclear hyperchromatism, stratification or atypia 10 Adenomatous nuclei are usually hyperchromatic, enlarged, cigar shaped and crowded together in palisade pattern10. Adenomas are classified as tubular or villous. Histologically, tubular adenomas are composed of branched tubules, whereas villous adenomas contain digitiform villi arranged in a frond. Tubulovillous adenomas contain both elements10. Virtually all colon cancers arise from adenomas as demonstrated by multiple epidemiologic, clinical and pathologic findings10.
The combination of molecular events that lead to colonic adenocarcinoma is heterogeneous and includes genetic and epigenetic abnormalities. At least two distinct genetic pathways have been described. In simplest terms, these are the APC/β-catenin pathway, which is associated with WNT and the classic adenoma-carcinoma sequence; and the microsatellite instability pathway, which is associated with defects in DNA mismatch repair. Both pathways involve the stepwise accumulation of multiple mutations, but the genes involved and the mechanisms by which the mutations accumulate differ. Epigenetic events, the most common of which is methylation-induced gene silencing, may enhance progression along both pathways.11
Colonoscopy is recommended for screening of patients more than 50 years old at average risk for colon cancer or colonic polyps10
Colonoscopy is a highly specific test. At colonoscopy polyps are removed and mass biopsied for pathologic diagnosis10.
The epidermal growth factor receptor (EGFR, cerbB-1) is the first identified member of the type I receptor tyrosine kinase family and is a major regulator of several diverse cellular pathways12.
The EGFR is a 170 kDa protein that consists of an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Gastric cancer is the 2nd most common cancer in the world, accounting for 9 % of all malignancies worldwide. Increased expression and activation of EGFR and its ligands are causally associated with progression of gastric cancer and poor prognosis13
While the majority of gastric cancers are not hereditary, the mutations identified in familial gastric cancer have provided important insights into mechanisms of carcinogenesis in sporadic cases. Germline mutations in CDH1, which encodes E-cadherin, a protein that contributes to epithelial intercellular adhesion, are associated with familial gastric cancers, which are usually of the diffuse type. Mutations in CDH1 are present in about 50% of sporadic cases of diffuse gastric tumors, while E-cadherin expression is drastically decreased in the rest, often by methylation of the CDH1 promoter. Thus, the loss of E-cadherin function seems to be a key step in the development of diffuse gastric cancer. Notably, CDH1 mutations are also common in sporadic and familial lobular carcinoma of the breast, which also tends to infiltrate as single cells, and individuals with BRCA2 mutations are at increased risk of developing diffuse gastric cancer14.
It has been well documented that over expression of EGFR in colon cancer may be linked to an advanced stage of the disease or may predict a potential metastatic risk15. EGFR has been found elevated in colorectal cancers, with expression rates ranging from 25 % to 77% 13. This heterogeneity of expression is attributed to the different detection techniques , although most are based on quantitative immunohistochemical labelling with monoclonal antibodies.13 Despite these known reproducibility and validation difficulties , immunohistochemical testing remains one of the most common and accurate methods to assess EGFR expression.16
In the Goldstein studyit is noteworthy that EGFR reactivity in the deepest region of the primary tumour had the strongest correlation with EGFR reactivity in lymph node and liver metastases15.
Blocking EGFR activation would obviously represent an innovative and key strategy in patient care because this therapeutic strategy impairs crucial functions linked to proliferation and survival. 17
ANNEXUREIII
6.3 AIMS AND OBJECTIVES
· To study the morphological lesions associated with gastric cancer and colorectal cancer.
· To study the positivity of epidermal growth factor receptor in all the biopsy using Hematoxylin &Eosin, and Immunohistochemistry staining techniques and their efficacy.
· To correlate epidermal growth factor receptor positivity with histological findings.
· To provide data which could be used as a prognostic indicator and which could be useful for subsequent treatment options.
7. MATERIALS AND METHODS
ANNEXURE IV
7.1 SOURCE OF DATA-
Study will be done in Pathology department of Dr .B R Ambedkar Medical College and Hospital in collaboration with Department of Surgery.
ANNEXURE V
7.2 METHOD OF COLLECTION-
Fifty cases of endoscopic and colorectal biopsies from June 2012 to June 2014 will be included in the study. These patients will have a clinical history of dyspeptic symptoms, bleeding per rectum, weight loss and anaemia.
Biopsies will be fixed in 10% formalin and histopathological slides will be prepared for staining with Haematoxylin and Eosin and Immunohistochemical methods using paraffin blocks.
INCLUSION CRITERIA
The study will include all biopsies which will be done for various chronic upper abdominal and lower abdominal symptoms such as abdominal pain, dyspepsia, heartburn, nausea, vomiting, bleeding per rectum, chronic diarrhoeas and also for associated systemic manifestations like anorexia, weight loss.
EXCLUSION CRITERIA- Acute symptoms and autolysed specimens.
STUDY SAMPLE DESIGN -Prospective and retrospective study
METHOD- Relevant statistical method will be used.
NUMBER OF CASES-A total of 50 patients with symptoms of dyspepsia, and lower abdominal symptoms such as diarrhoea, bleeding per rectum will be selected for the study.
ANNEXURE VI
7.3.STAINING TECHNIQUE
H&E stained sections of all the cases will be reviewed. All endoscopic gastric mucosal biopsies taken from different sites will be brought in 10% buffered formalin and will be oriented with submucosa embedded downwards. After overnight fixation in formalin, dehydration done with graded alcohol, clearing in chloroform followed by paraffin embedding and section cutting in rotary microtome. Sections of 3 μm thicknesses will be made & stained with H&E and immunohistochemical stains. Morphological changes in all cases will be studied with special reference to depth of invasion, the presence or absence of lymph node metastases.
HEMATOXYLIN and EOSIN18 - Procedure:
1. Dewax sections, hydrate through graded alcohol to water
2. Stain in alum haematoxylin for 5 minutes.
3. Wash in running tap water until sections blue for 5 minutes or less
4. Differentiate in 1%acid alcohol for 5-10 seconds
5. Wash well in tap water until sections are again blue
6. Blue by dipping in an alkaline solution (ammonia water) followed by a 5 min tap water wash
7. Counter stain in 1% eosin Y for 30 seconds to 1 minute.
8. .Dehydrate, clear and mount in DPX
Result: The nuclear component stains blue (basophilic) and the cytoplasmic component stains pink (eosinophilic).
IMMUNOHISTOCHEMISTRY18
1. Formalin fixed paraffin embedded sections are taken on PLL quoted slides.