EVALUATION OF PROGNOSTIC INDICATORS IN COLORECTAL CARCINOMA – A COMPREHENSIVE STUDY TO CORRELATE STAGING, GRADING AND PROLIFERATIVE INDICATORS
DISSERTATION
SUBMITTED TO TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY,
CHENNAI
in partial fulfilment of
the requirements for the degree of
M.D. (PATHOLOGY) BRANCH - III
TIRUNELVELI MEDICAL COLLEGE HOSPITAL,
TIRUNELVELI- 627011
MAY 2019 CERTIFICATE
I hereby certify that this dissertation entitled “EVALUATION OF
PROGNOSTIC INDICATORS IN COLORECTAL CARCINOMA – A
COMPREHENSIVE STUDY TO CORRELATE STAGING, GRADING AND
PROLIFERATIVE INDICATORS” is a record of work done by
Dr.P.CHINTHU, in the Department of Pathology, Tirunelveli Medical College,
Tirunelveli, during her postgraduate degree course period from 2016- 2019. This work has not formed the basis for previous award of any degree.
Dr. S.M. KANNAN M.S.,M.Ch; DEAN Tirunelveli Medical College, Tirunelveli - 627011. CERTIFICATE
I hereby certify that this dissertation entitled “EVALUATION OF PROGNOSTIC
INDICATORS IN COLORECTAL CARCINOMA – A COMPREHENSIVE STUDY
TO CORRELATE STAGING, GRADING AND PROLIFERATIVE INDICATORS” is a record of work done by Dr.P.CHINTHU, in the Department of Pathology,
Tirunelveli Medical College, Tirunelveli, during her postgraduate degree course period from 2016- 2019,under my guidance and supervision, in the Department of
Pathology Tirunelveli Medical College & Hospital, Tirunelveli, in partial fulfilment of the requirement for M.D., (Branch III) in Pathology examination of the Tamilnadu
Dr. M.G.R Medical University to be held in MAY 2019. This work has not formed the basis for previous award of any degree.
Prof. J. SURESH DURAI M.D., Department of pathology, Tirunelveli Medical College, Tirunelveli- 627011.
Prof. K. SHANTARAMAN M.D., Professor and Head, Department of Pathology, Tirunelveli Medical College Tirunelveli- 627011.
. DECLARATION
I solemnly declare that this dissertation titled “EVALUATION OF PROGNOSTIC
INDICATORS IN COLORECTAL CARCINOMA – A COMPREHENSIVE STUDY
TO CORRELATE STAGING, GRADING AND PROLIFERATIVE INDICATORS” submitted by me for the degree of M.D, is the record work carried out by me during the period of 2016-2019 under the guidance of Dr. J. SURESH DURAI, M.D,
Professor of Pathology, Department of Pathology, Tirunelveli Medical College,
Tirunelveli. The dissertation is submitted to The Tamilnadu Dr. M.G.R. Medical
University, Chennai, towards the partial fulfilment of requirements for the award of
M.D. Degree (Branch III) Pathology examination to be held in May 2019.
Place: Tirunelveli Dr.P.CHINTHU, Postgraduate, Date: Department of Pathology, Tirunelveli Medical College, Tirunelveli-11. ACKNOWLEDGEMENT This dissertation has come to your hands as a result of the combined effort of lot of people. I am thankful to all these people for helping me in bringing out this work.
I thank the DEAN Dr.S.M.Kannan M.S;M.Ch; for permitting me to conduct this study and to avail the resources of the hospital.
I am also thankful to my Professor and Head, Department of Pathology
DR.K. SHANTARAMAN M.D, for his support and guidance in doing this dissertation.
I express my heartfelt gratitude to my guide DR. J. SURESH DURAI M.D,
Professor, Department of Pathology, who has guided me through all the steps in my study right from the beginning till the end.
I am extremely thankful to my PROFESSORS DR. K. SWAMINATHAN
M.D., DR.ARASI RAJESH M.D., DR.VASUKI M.D,
DR.V.BHAGIYALAKSHMI M.D, Department of Pathology, for guiding me during the period of study.
I am extremely thankful to Assistant Professors, Dr.Johnsy Merla M.D,
Dr.Hidhaya Fathima M.D, Dr.Sindhuja M.D, Dr.Mahalakshmi M.D, Dr.Dina
Mary MD, Dr. Dharma saranya MD, Dr.Chandhru Mari MD, Dr.Selin sofia
MD, Dr.Gurusamy MD, Dr.Ramasundari illambirai MD, Department of
Pathology, for guiding me academically and professionally during the period of study.
I also thank the lab technicians Mrs.Veeralakshmi, Mr.Sankaranarayanan,
Mr.Balamurugan, Mrs.Amutha, Mrs. Sivanadiyal, Mrs.Mahalakshmi and Mr.Balachander for their support and cooperation which enormously helped me in the study. Without their humble cooperation, this study would not have been possible.
I also thank my fellow postgraduates and my friends for their support.
I thank GOD AND MY FAMILY, for blessing me not only in this study, but in all endeavours of my life.
CERTIFICATE – II
This is certify that this dissertation work title “EVALUATION OF
PROGNOSTIC INDICATORS IN COLORECTAL CARCINOMA – A
COMPREHENSIVE STUDY TO CORRELATE STAGING, GRADING AND
PROLIFERATIVE INDICATORS” of the candidate Dr.P.CHINTHU, with registration Number 201613303 for the award of M.D. Degree in the branch of
PATHOLOGY (III). I personally verified the urkund.com website for the purpose of plagiarism check. I found that the uploaded thesis file contains from introduction to conclusion page and result shows 9 percentage of plagiarism in the dissertation.
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CONTENTS
S.NO TITLE PAGE.NO
1. INTRODUCTION 1
2. AIM AND OBJECTIVES 3
3. REVIEW OF LITERATURE 4
4. MATERIALS AND METHODS 52
5. OBSERVATION AND RESULTS 58
6. DISCUSSION 79
7. SUMMARY 86
8. CONCLUSION 88
BIBILIOGRAPHY
MASTER CHART INTRODUCTION
Colorectal cancer (CRC) is the third most common cancer worldwide with a high mortality at the advanced stages1. Many risk factors such as advanced age, male sex, family history, dietary factors such as diets rich in red meat, fat and low in fruits and vegetables, life style factors such as obesity, smoking , alcohol play a major role in the pathogenesis of colorectal carcinoma. With increasing westernization, the incidence of colorectal carcinoma has been increasing in previously low incidence countries, including India2,3.
Colorectal carcinoma is a heterogeneous disease. Three major molecular pathways have been identified leading to colorectal carcinogenesis. They are (1) Chromosomal instability (CIN) pathway, accounting for upto 85% of CRC (ii) Microsatellite instability (MSI) pathway (iii) CpG island methylator phenotype (CIMP) pathway4.
Patients with familial colorectal carcinomas require both diagnostic testing and molecular screening5.
Almost 85% of the colorectal cancers occur sporadically and about 15-20% are inherited6. Colorectal cancers usually occurs from the pre-existing adenomas, hence the disease can be prevented and curable if diagonosis is made at the early stages. Hence screening with colonoscopy, flexible sigmoidoscopy and occult blood test are essential7 .Colonoscopic
1 biopsy and pathological examination remains the gold standard in the diagnosis of colorectal carcinoma. Imaging aids in the proper staging prior to surgery5.
The prognosis in each individual case is obtained through the histopathological confirmation of the adequacy of excision, tumour stage, type and grade. Currently Tumour, Node and Metastasis (TNM) staging system provides an almost accurate prognostic information in colorectal carcinoma. The prognosis also varies with tumour type and grade.
However, there is a heterogeneity in the clinical features and survival rates among the patients with the same stage. Hence the application of immunohisto-chemical studies with proliferation markers such as Ki67, provides a step ahead in establishing the prognosis.
2 AIMS & OBJECTIVE
The study is aimed at
1. Staging of different grades of colorectal carcinomas and evaluating
the role of proliferative immunomarkers in each stage.
2. Predicting the tumour aggressiveness, biological behaviour and
prognosticating the disease outcome using specific cell proliferation
indicators.
3 REVIEW OF LITERATURE
HISTORY
The sentinel account of a hereditary colorectal family was discovered by Dr. Aldred Warthin, who first suspected the disorder in the family of an affected woman (who subsequently died of endometrial cancer) over 100 years ago. He began studying her family (Family G) in
1895 and published his first report on it in 19138. In 1971, updated studies of Family G by Lynch and Krush showed it to be consonant with what became known as Lynch syndrome9.
Knudson’s two hit hypothesis provided the basis of our understanding of how tumor suppressor genes could explain the younger ages of onset in familial cancers as well as variable penetrance10. Although susceptibility is increased, a second mutation is required to produce a tumor. Fearon & Vogelstein showed us that in some cancers, the adenomatous polyposis coli (APC) gene is mutated as the initial step in the carcinogenic pathway11 .
4 CLINICAL PRESENTATION
Colorectal carcinoma is usually the disease of aged 65-74, with higher prevalence in women12. Now due to changing lifestyles such as sedentarism , obesity, bad nutritional habits such as diet rich in fat and protein, smoking, etc, its frequency has been increasing in the younger population.
The clinical features includes symptoms like abdominal pain, alterations of bowel habits, weight loss, nausea, vomiting, fatigue, loss of appetite and abdmimal distension. Usually distal cancers causes rectal bleeding compared to proximal cancers which usually presents with anaemia due to occult blood loss13.
EPIDEMIOLOGY
In the report of GLOBOCAN 2012, colorectal cancer had been reported as the third most common in men with 1361,000 cases representing about 10% of all the cancers and it has been the second most common type of cancer in women with 614,00 cases representing 9.2% of all the cancers in the United states14. With the use of colonoscopy screening methods, a gradual decrease in incidence was observed which indicates the early detection with removal of precancerous lesions in adults
5 from 50 to 75 years of age15,16. The use of colonoscopy has been increased from 19.1% to 54%5 in 201317.
FUNCTIONAL ANATOMY
The colon and rectum forms the major portion of the large intestine which extends from the end of ileum to the anus. It is about 1.5 meters long which makes about one-fifth of the entire intestinal canal. The colon is divided into four parts : the ascending colon, transverse colon, descending colon and sigmoid colon.
The large intestine has four coats : serous, muscular, areolar
(submucous) and mucous. The serous coat is derived from the peritoneum, which covers the large intestine in variable extents . The caecum, transverse colon and sigmoid colon are completely surrounded by serous covering. The ascending colon has covering only in the front and at the sides. The rectum is covered in its upper part on the anterior surface and its sides, and lower part on its anterior surface only. The muscular coat consists of outer longitudinal and inner circular layers. The areolar coat connects the muscular layer with mucous layer. The mucous layer of the large intestine has glands lined by short columnar epithelium which has numerous goblet cells. The mucous layer also has solitary lymphatic nodules and a muscularis mucosa layer 18.
6 AETIOLOGY AND RISK FACTORS
The etiology of colorectal carcinoma is very complex and it involves the interactions between multiple factors such as
Age
Sex
Life-style
Chronic inflammation
Environmental factors and
Genetic factors
AGE
About 90% of the colorectal carcinoma occurs in the age group of
50 and above. The incidence increases in the age group of 60 to 79.
However, now colorectal carcinoma is one among the 10 commonly diagnosed cancers in the age of 20 to 49 years19.
DIET
Diet has the strong influence in the development of colorectal carcinoma Diets rich in fat favors the increased production of bacterial flora which degrades bile salts to potentially carcinogenic N-nitroso compounds . High consumption of red meat is also implicated in the development of colorectal carcinoma. The high content of heme iron in red
7 meat under high temperatures results in the production of heterocyclic amines and polycyclic aromatic hydrocarbons which are carcinogenic20 .
Also diet low in fruits and vegetables, reduced dietry fibre intake can lead to risk of development of colorectal carcinoma.
LIFE-STYLE
Physical inactivity and obesity are two modifiable risk factors linked to colorectal carcinoma21. Heavy alcohol consumption and cigarette smoking has been associated with the onset of colorectal cancer at the younger age22. The risk in alcoholics is further enhanced when there is a phenotypic loss of Aldehyde dehydrogenase 2, and loss of heterozygosity of mthfr [5,10-Methylene Tetrahydrofolate Reductase].
CHRONIC INFLAMMATION
Chronic inflammation caused by inflammatory bowel disease may be contributory factor in the pathogenesis of colon cancer. Patients with ulcerative colitis have an increased risk . KLF 6 (kruppel like factor 6), a ubiquitous zinc finger tumour suppressor is inactivated by loss or mutation in most of the colorectal carcinomas occuring on the basis of inflammatory bowel disease23.
8 ENVIRONMENTAL FACTORS
Colon plays a major role in digesting the exogeneous carcinogens which reaches the colon. So the action of many metabolic enzymes are of much importance in the detoxification of these carcinogens.
Chemical carcinogens that are metabolically activated bind to the
DNA which may result in cancer . Cytochrome P450 1A2 is involved in the metabolic activation of polycyclic aromatic hydrocarbons which are associated with an increased risk of colon cancer. Other enzymes such as
Cytosolic glutathione S-transferases, Arylamine N-Acetyl Transferase are also involved in the activation22. Persons who are rapid acetylators and have high cytochrome P450 1A2 activity tend to have an increased risk for developing colorectal carcinoma.
GENETIC FACTORS
Many hereditary cancer syndromes such as Juvenile polyposis,
Familial Adenomatous Polyposis, Gardner Syndrome , Turcot Syndrome,
Hereditary Non-Polyposis Colorectal Cancer, MYH-Adenomatous
Polyposis Syndrome, Peutz- Jeghers Syndrome, Birt Hogg Dube
Syndrome, Cowden syndrome have been associated with colorectal carcinoma.
9 Juvenile Polyposis
Juvenile Polyposis is an autosomal dominant syndrome. These patients have about 50-200 polyps mostly involving the rectosigmoid region. The polyps contain multiple cystic spaces, lined by columnar epithelium surrounded by abundant stroma. These patients have inactivating mutations in the gene that encodes BMP (bone morphogenetic protein) receptor 1A . Cases with early onset at the age of
3-5 have mutations in edg (endoglin). Endoglin is a homodimeric transmembrane glycoprotein and a part of TGF-β complex which contributes to angiogenesis and aids in the development of polyps.
Hemorrhagic teleangiectasias occur in this syndrome when associated with germline mutations of smad 4 gene located in the chromosome 18q21.1.
Individuals with juvenile polyposis syndrome have a great risk for developing colorectal carcinoma24 .
Familial Adenomatous Polyposis ( FAP )
Familial Adenomatous Polyposis is an autosomal dominant condition in which colorectal carcinoma occurs in early adulthood secondary to numerous adenomatous polyps in colon. FAP is due to the mutations of the APC gene located in the chromosome 5q21. Most mutations accumulate in the central region, described as mutation cluster region (MCR) which result in the expression of a COOH-terminally
10 truncated protein. FAP occurs in conjunction with other extracolonic tumours such as brain cancer, thyroid cancer, pancreatoblastoma, hepatocellular adenoma, adrenocortical adenoma or uterine adenocarcinoma. Congenital hypertrophy of retinal pigment epithelium is also noted in many cases24.
Gardner Syndrome
Gardner Syndrome is a phenotypic variant of FAP . It consists of familial adenomatous polyposis in association with other extracolonic abnormalities like osteomata, epidermoid cysts and intraperitoneal desmoid tumours. Gardner syndrome also involves defect in the APC gene24.
Turcot Syndrome
Turcot Syndrome is a rare autosomal recessive condition, associated with primary neuroepithelial tumours of the central nervous system and numerous adenomatous colorectal polyps. Patients with turcot syndrome developing medulloblastoma usually have germline APC mutations24.
Hereditary Non-Polyposis Colorectal Cancer (HNPCC)
HNPCC mostly involves the proximal colon. The average age of onset is 45 years. HNPCC is associated with microsatellite instability.
There occurs germline loss of function mutations in DNA mismatch repair
11 genes msh 2, mlh 1, msh 3and msh 6 .These tumours are predominantly right sided and mostly poorly differentiated. HNPCC is subdivided into 3 clinical forms- Lynch type1, Lynch type 2 and Muir – Torre syndrome. In
Lynch type 1 syndrome, patients have only colorectal manifestations .
Lynch type 2 syndrome is associated with cancers involving ovary, endometrium, hepatobiliary tract, small bowel, renal pelvis and ureter.
Muir-Torre syndrome is associated with sebaceous adenomas and keratoacanthomata24.
MYH- Adenomatous Polyposis Syndrome
MYH-Adenomatous Polyposis Syndrome is an autosomal recessive disorder with multiple polyps involving colon and rectum. This is caused by homozygous mutations in the base excision repair gene myh (mutant
YH)24.
Peutz- Jeghers Syndrome
Peutz- Jeghers Syndrome is an autosomal dominant condition caused by mutations in the stk 11 (serine / threonine kinase 11) gene.
Patients have melanocytic macules of the lips, buccal mucosa and digits and associated with multiple gastrointestinal hamartomatous polyps24.
12 Birt Hogg Dube Syndrome
Birt Hogg Dube syndrome is associated with risk for intestinal polyposis.This is a rare inherited genodermatosis associated with hair follicle hamartomata, kidney tumours and spontaneous pneumothorax.
This syndrome is otherwise called as fibrofolliculomata with trichodiscomata and acrochordans24 . It is caused by mutations in folliculin gene located in the chromosome 17p11.2.
Other Syndromes
Cowden syndrome, Bannayan-Riley-Ruvalcaba syndrome,
Lhermitte -Duclos disease and Proteus syndrome are associated with hyperplastic and dysplastic changes in the skin , colon and prostate. These are due to germline pten (10q23.31) mutations24.
COLORECTAL CARCINOGENESIS
The normal adult colon epithelium consists of three differentiated cell types that arise from a multipotent stem cell: absorptive epithelial cells, enteroendocrine cells and goblet cells. The intiation for neoplastic transformation is likely to occur in stem cells or their early descendants which leads to deviation from the normal maturation pathway. Many sequential genetic changes need to occur in the evolution of colorectal carcinoma. Colorectal tumours undergo a sequence arising from normal
13 epithelium through more severe degrees of dysplasia from aberrant crypt foci through adenoma to carcinoma.
FIGURE 1: PROGRESSION OF THE ADENOMA TO
CARCINOMA SEQUENCE .
Majority of the cases of colorectal carcinomas occur sporadically, only 5-10% of the colorectal carcinomas are due to inherited mutations in cancer-related genes25. The mechanisms involved in the initiation and development of colorectal carcinoma demonstrates the existence of three major molecular pathways :
The Chromosomal instability (CIN) pathway
CpG island methylator phenotype (CIMP) pathway
Microsatellite Instability (MSI) pathway
14 Colorectal carcinomas developing through the CIN pathway and
in HNPCC,mostly originate from adenomatous polyps. Colorectal
carcinomas developing through the CIMP pathway has the sessile
serrated adenomas as the chief pathological precursors27. Recently
microRNAs and inflammation have been attributed to the causation of
colorectal carcinoma. Several genetic and epigenetic changes are
important in determining patient prognosis and survival.
1.CHROMOSOMAL INSTABILITY (CIN)
Chromosomal instability is the most common form of genomic instability found in 70-85% of the colorectal carcinomas . Chromosomal instability is defined as the presence of numerical chromosome changes or multiple structural aberrations of the chromosomes, which are recognized by the presence of aneuploidy or polyploidy26. This includes gene deletions, duplications and chromosomal rearrangements. These can be assessed by a variety of techniques such as DNA flow cytometry, comparative genomic hybridization, whole exome sequencing and high density SNP arrays. Chromosomal abnormalities have been detected in colon adenomas, suggesting CIN may arise early in the polyp→ colorectal carcinoma sequence.
The earliest identifiable lesion in this pathway is the dysplastic aberrant crypt focus (ACF) which is a microscopic mucosal
15 lesion that precedes the development of a polyp. The CIN pathway is associated with mutation in APC gene or loss of chromosome 5q that includes the APC gene, mutation of the KRAS oncogene, loss of chromosome 18q and deletion of chromosome 17p, which contains the tumor suppressor gene TP53. APC is an important tumour suppressor gene in the CIN pathway to colorectal carcinoma. It is the “key” initial mutation involved in both sporadic CIN and all germline mutated FAP27.
The most commonly involved pathways included in the colorectal carcinogenesis under chromosomal instability are:
WNT Signaling Pathway ,
RAS Pathway,
P53 Pathway
The WNT Signaling Pathway
The gastrointestinal epithelium contains crypts and villi. Cell division occurs in the crypts and the cells gradually move up the crypt walls to the villi as they differentiate. WNT signaling maintains the crypt progenitor compartments and controls the cell fate during differentiation along the crypt-to-villus axis .
The binding of APC to β-catenin helps to suppress the Wnt- signalling pathway. Mutations in APC produce a truncated APC protein,
16 thereby interrupting the binding of APC to β-catenin. The β-catenin cytoplasmic accumulation leads to its translocation into the nucleus and stimulates the TCF- targets, with increased proliferation, differentiation, migration and adhesion of colorectal cells. Mutations in β-catenin without
APC mutations are also present in the early stages of the colorectal pathogenesis and possibly substitutes the APC mutations in the stages of initiation28. CDK8 (cyclin-dependent kinase-8) gene located at 13q 12.13 acts as an oncogene in approximately 60% of colorectal carcinoma cases by stimulating both β-catenin and Notch 1, thus increasing transcription and cell differentiation29.
Recently, activation of orphan receptors LGR-4 and LGR-5
(leucine-rich repeat G- protein coupled Receptor), G-protein coupled receptors, was found to increase signaling by binding with proteins in the
R-respondin family, known potentiators of the WNT signaling pathway30.
Cyclin D1 (CCND1) together with other cyclin dependent kinases that inhibit cyclin D1 such as p27 (CDKN1B) and p21 (CDKN1A) are central to cell cycle control - mainly in the transistion from G1 to S phase.
Excessive CCND1 activation by APC mutation contributes to the development of colonic neoplasia by allowing the cell to evade apoptosis31.
17 RAS Pathway
In over 40% of colon cancers, activation of ras oncogenes by point mutations (never H-ras, rarely N-ras and mostly K-ras) is the causative. K- ras(12p12) encodes a GTP-binding protein involved in the transduction and propagation of extracellular signals, which when mutated, can cause a loss of inherent GTPase activity and thus constitutively signaling occurs through the downstream , RAS-RAF-MEK-ERK pathway. In K-ras, the activation typically occurs by a change in the coding properties of codons
12 or 1333. Codon 61 may also be affected. A G12V (glycine to valine at codon 12) mutation of RAS is associated with aggressive disease and high risk of recurrence. Mutations of K-ras leads to a permanently active state that permits the cell to evade apoptosis and acquire a growth advantage.
FGFR signals through RAS. Activating mutations in the fgfr 3 gene can cause excessive RAS activity and are associated with colorectal cancer32.
P53 Pathway
P53 is a tumour suppressor gene located at the chromosome 17p.
The function of P53 protein is to increase the expression of cell cycle genes, to slow the cell cycle and provide sufficient time for DNA repair.
Loss of chromosome 17p, associated with missense mutations in the remaining p53 allele, occurs in about 75% of colorectal carcinoma, but infrequently in benign lesions, indicating that loss of p53 is involved in the
18 later stages of colorectal carcinogenesis. Mutation in p53 stimulates high proliferative activity through the loss of cell cycle control and apoptosis34.
Mutations in five hot spots (codons 175,248,282,273,245) account for over
40% of p53 mutations in colorectal cancer. CT transitions at CpG sites are the most prevalent mutations in the p53 gene in colon tumours and in the germline Li-Fraumeni syndrome. P53 also interacts with cycloxygenase -2, which plays a role in promoting inflammation and cell proliferation in colorectal carcinoma35.
Other pathways involved in chromosomal Instability:
A mutation occurring simultaneously with APC gene mutations is the mutation in the PI3KCA gene that stimulates cell growth and production of fatty acid synthase through the AKT pathway. PI3KCA mutations also interact with a central regular of cell growth and metabolism, mTOR and with K-ras36,37.
A molecular alteration often co-occuring with the p53 loss is the loss of chromosome 18q, where the genes smad 2, smad 4 and DCC gene are located. Loss of 18q is associated with a strong negative prognosis in colon cancer, in view of high metastatic potential.
LINE – 1 (Long Interspersed nucleotide element -1) retrotransposon has been associated with chromosomal instability in colorectal carcinoma
19 through global DNA hypomethylation which in turn leads to the expression of non –coding RNA. Researchers have found out that this pathway is present in the colorectal carcinoma patients with young age of onset (<60 years) and has a more aggressive behavior38,39.
Other CIN Pathway which contributes to aneuploidy in the early stage of tumorigenesis is represented by the alterations of the kinetochore, a multiprotein complex essential for chromosome segregation during mitosis. Over expression of Centeromere protein (CENP) –A and CENP –
H, the two kinetochore components have been found in many cases of colorectal carcinoma . This overexpression causes an altered association with non-centeromeric regions of chromatin, resulting in the disruptions of the kinetochore complex40.
Hypoxia Inducible Factor (HIF) – 1 and HIF-2 are the other components in the CIN pathway. They act as essential mediators of cellular response to hypoxia and elevate the expression of many genes involved in angiogenesis, glucose metabolism and cell survival through different pathways, including mTOR. Overexpression of a subunit of HIF
1 and HIF 2, namely HIF1α directly upregulates COX-2 expression in colorectal carcinoma by binding to it. This HIF1α overexpression has been associated with shorter colorectal cancer-specific survival41.
20 The expression of cathepsin – B ( CTSB ), a lysosomal cysteine protease, has been found elevated in all stages of colorectal carcinoma from early tumour initiation to the metastatic lesions. It has been strongly associated with increase in risk of colorectal carcinoma - specific mortality42.
2.CpG ISLAND METHYLATOR PHENOTYPE(CIMP) PATHWAY
CIMP is found in approximately 20-30% of colorectal carcinomas43.
Tumours of the CpG island methylator phenotype have a high degree of
CpG island hypermethylation in tumour suppressor genes or in DNA repair genes such as p16 and MLH1. The promoter hypermethylation is often a result of mutations in K-ras and TGF-βR-II. The loss of TGF-β control is the initial defect in the CpG island methylator phenotype .
The classic panel for CIMP determination consists of CpG sites in p16, MLH1 and methylated in tumours (MINTs) 1,2 and 31. CIMP- positive tumours are divided into two types:(i) CIMP-high, related ot
BRAF mutations and MLH1 methylation (ii) CIMP-low, related to KRAS mutations and microsatellite stability. CIMP negative tumours are microsatellite stable with frequent TP53 mutation.Systemic screening of
195 CpG sites including neurogenin (NEUROG1), IGF 2, runt-related transcription factor 3 (RUN x3) and suppressor of cytokine signalling 1
(SOCS 1) provided an alternative to classic panel. This do not have a
21 relationship to K-RAS but is strongly associated with BRAF V600E mutation25.
3. MICROSATELLITE INSTABILITY(MSI) PATHWAY
Microsatellite instability pathway is involved in the genesis of approximately 15% of sporadic colorectal carcinomas and >95% of
HNPCC syndromes. Microsatellites are nucleotide repeat sequences scattered through out the genome. Microsatellite instability refers to the presence of atleast 30% unstable microsatellite loci in a panel of 5-10 loci consisting of mono- and di-nucleotide tracts. Tumours with only 10-29% unstable loci has been designated as having “MSI – low”26 . DNA polymerase is mostly susceptible for making errors when copying these short repeat sequences and hence mismatch repair dysfunction results in
MSI.
The Mismatch Repair (MMR) system is composed of seven proteins namely MLH1,MLH 3, MSH 2, MSH 3, MSH 6, PMS 1 and PMS
2, which in association with specific partners forms functional heterodimers. The essential functional heterodimeric proteins include MSH
2-MSH 3, MSH 2 - MSH 6,MLH 1 - PMS1, MLH 1 –PMS 2, MLH1-
MLH 327.
22 The mechanisms underlying MSI involve inactivation of genes in the DNA mismatch repair (MMR) family either by aberrant DNA methylation or by somatic mutation. Individuals with HNPCC (Lynch syndrome) develop MSI colorectal carcinomas by the germline mutations in one of the MMR genes, MLH 1, MSH 2, MSH 6 and PMS 2. On the other hand, sporadic MSI colorectal carcinomas develop as a result of loss of MMR activity by aberrant DNA methylation leading to silencing of
MLH 126 . Sporadic MSI – high colorectal carcinoma cases harbor the
V600E mutation of the BRAF oncogene, a member of the RAF family involved in the mediation of the cellular response to the growth signal through the RAS-RAF-MAP kinase pathway.
Colorectal carcinomas developing through MSI pathway are usually located in the proximal colon, often associated with a poorly differentiated and a mucinous or medullary histology and has intense peritumoral and intratumoral lymphocytic infiltrations. Patients with MSI-high colorectal carcinoma have a better prognosis and longer survival than the patients with chromosomal instability positive colorectal carcinoma43.
23 FIGURE 2: PATHWAYS OF COLORECTAL CARCINOGENESIS
MICRO RNA (miRNA)
miRNAs are a class of short (20-25 nucleotide) non-coding
RNAs.They regulate the protein expression by the inhibition of mRNA translation of genes involved in cell differentiation, development, proliferation and apoptosis. Many miRNAs have been involved in the pathogenesis of colorectal carcinoma, the number is still expanding as many new miRNAs are being identified. They can be upregulated or downregulated, operating like oncogenes and tumour suppressor genes44.
24 Lanza et al. found out that the upregulation of miR -17-92, miR-17-
5p, miR-20, miR25, miR-92-1, miR-98-1, and miR-106a are found in microsatellite stable colorectal carcinoma and not in MSI-colorectal carcinoma53. Motoyama et al. have shown that the overexpression of miR-
18a is associated with worse colorectal carcinoma prognosis and expression of miR-21 has a poor prognosis in patients with stage IV cancer52.
INFLAMMATORY PATHWAY
There has been a strong association between colorectal carcinoma and inflammatory bowel disease and chronic NSAID use , thereby chronic inflammation playing a critical role in colorectal carcinoma initiation and progression. Mechanisms by which chronic inflammation leads to carcinogenesis include sustained cell growth by the anti-apoptotic system, increased DNA damage through the activation of the mutagenic reactive oxygen and nitrogen species,increased production of angiogenic and lymphangiogenic growth factors and changes in the membrane systems to facilitate invasion and altering cell adhesion.
Chronically elevated levels of the pro-inflammatory cytokine TNF
- α promote tumour growth. IL-6 is a cytokine involved in the acute phase of inflammation ,stimulates the transcription of STAT 345. STAT 3 activation stimulates the cell proliferation, differentiation apoptosis and
25 promotes metastasis by inducing the expression of different gene targets - such as VEGFR - 2, BCl-2, Cyclin D1,MMP 2-9, ICAM -1 and COX -2.
BETHESDA TESTING GUIDELINES46
Below are the Revised Bethesda Guidelines for testing colorectal tumors for microsatellite instability (MSI):
Colorectal cancer diagnosed in a patient who is less than 50 years
of age.
Presence of synchronous, metachronous colorectal, or other HNPCC
associated tumours regardless of age
Colorectal cancer with the MSI – H histology diagnosed in a patient
who is less than 60 years of age.
Colorectal cancer diagnosed in one or more first degree relatives
with an HNPCC - related tumour with one of the cancer being
diagnosed under 50 years of age.
Colorectal cancer diagnosed in two or more first or second - degree
relatives with HNPCC related tumours, regardless of age.
Further testing of colorectal tumours such as IHC for MLH 1, MSH 2,
MSH 6 and PMS -2 and Microsatellite instablility are required in the above circumstances.
26 WHO CLASSIFICATION OF COLORECTAL CARCINOMAS47
The WHO classification of Carcinomas of colon and rectum, currently on the 4th edition published in 2010 are as follows:
I. Adenocarcinoma
a) Cribriform comedotype adenocarcinoma
b) Medullary carcinoma
c) Micropapillary carcinoma
d) Mucinous adenocarcinoma
e) Serrated adenocarcinoma
f) Signet ring cell carcinoma
II. Adenosquamous carcinoma
III .Spindle cell carcinoma
V. Undifferentiated carcinoma
The term ‘carcinoma’ is used for tumours of the colon and rectum when they invade through the muscularis mucosa into the submucosa.
More than 90% of the colorectal carcinomas are adenocarcinomas.
27 Macroscopy :
Colorectal carcinomas can present with variable macroscopic appearances48. They are :
1. Exophytic / Fungating with intraluminal growth
2. Endophytic / ulcerative with intramural growth
3. Annular / circumferential involvement of the colorectal wall with
luminal stenosis
4. Diffuse Infiltrative / Linitis plastica pattern which is the least
common.
They can be an overlap among these features and ulceration
is common. Mostly carcinomas proximal to the splenic flexure
present as exophytic masses while those in the descending colon and
rectum are mostly endophytic and annular. The cut surface of most
CRCs have a homogenously grayish white appearance, there can be
gelatinous areas in mucinous tumours.
Histopathology :
ADENOCARCINOMA
Adenocarcinoma form about 90% of the colorectal carcinomas47.
Most adenocarcinomas are gland-forming with varying configuration and size of the glandular structures. In well and moderately differentiated
28 carcinomas, the epithelial cells are tall and large, the gland lumina may have cellular debris. The varients of adenocarcinoma are:
(i)Mucinous Adenocarcinoma
The adenocarcinoma is termed mucinous if >50% of the lesion is composed of pools of extracellular mucin with malignant epithelium arranged as acinar structures, layers of tumour cells or individual tumour cells including signet-ring cells. Most of the mucinous adenocarcinomas are MSI-H ,are therefore low grade. Mucinous adenocarcinomas which are microsatellite stable or MSI-L behave as high grade lesions. If the mucinous areas are <50% in the carcinoma it is termed as adenocarcinoma having a mucinous component48.
(ii) Signet ring cell carcinoma
Adenocarcinoma is termed as signet ring cell carcinoma when >50% of the tumour cells have prominent intracytoplasmic mucin with typical displacement and moulding of the nucleus. Signet-ring cells can also be found in the pools of mucinous adenocarcinoma. Large signet ring cells are called as “globoid cells”. Some signet ring cell carcinomas are MSI-H and they behave as low grade lesions, but those lacking MSH-H behave in an aggressive manner. Carcinomas having signet ring cells areas <50% are termed as adenocarcinomas with a signet-ring cell component48.
29 (iii) Cribriform comedo-type adenocarcinoma
Cribriform comedo-type adenocarcinoma has extensive areas of cribriform glands with central necrosis. These tumours are usually microsatellite stable with CpG island hypermethylation47.
(iv) Medullary carcinoma
Medullary carcinoma has sheets of malignant cells with abundant eosinophilic cytoplasm, vesicular nucleus and prominent nucleoli with an extensive infiltration by intraepithelial lymphocytes. Most medullary carcinomas have MSI-H and have a favourable prognosis48.
(v) Micropapillary adenocarcinoma
Micropapillary adenocarcinoma is composed of small clusters of tumour cells within the stromal spaces mimicking vascular channels. This pattern can also be present in conventional colorectal carcinoma. This varient has a characteristic MUC1 staining pattern in immunohistochemistry47.
(vi) Serrated adenocarcinoma
Serrated adenocarcinoma has the architectural similiarity resembling a sessile serrated polyp with glandular serration that can be accompanied by mucinous, cribriform, trabecular and lacy areas with a low nucleus to
30 cytoplasm ratio. These tumours can have MSI-L or MSI-H, BRAF mutations, and CpG island hypermethylation47.
Adenosquamous carcinoma
Adenosquamous carcinoma shares the features of both adenocarcinoma and squamous cell carcinoma, either as seperate areas within the tumour or as intermingled areas. Pure squamous cell carcinoma is very rare47.
Spindle cell carcinoma
Spindle cell carcinoma is a biphasic carcinoma with a spindle cell sarcomatoid component. Here the tumour cells show focal immunoreactivity to keratins47.
Undifferentiated carcinoma
Undifferentiated carcinomas are very rare tumours lacking the morphological, immunohistochemical and molecular biological evidence of differentiation with that of an epithelial tumours and have variable histological features. Some of these tumours can have MSI-H47.
31 HISTOLOGICAL GRADING OF COLORECTAL
ADENOCARCINOMAS
Adenocarcinomas are graded according to the degree of tubule formation and cellular array in tumor tissue as follows:
Grade 1 or Well differentiated : >95% with gland formation
Grade 2 or Moderately differentiated : 50-95% with gland formation
Grade 3 or Poorly differentiated : 0-49% with gland formation
Grade 4 or undifferentiated carcinoma has now been reserved for
malignant epithelial tumours with no gland formation, mucin
production, neuro-endocrine, squamous or sarcomatoid
differentiation.
This morphological grading of tumours applies only to
Adenocarcinoma, NOS. Other morphological varients carry their own prognostic significance and this grading cannot be applied for them47,48.
32 STAGING
HISTORICAL STAGING SYSTEMS
DUKES STAGING:
The classification proposed by C.Dukes in 1929-35 for rectal cancer served as the template for many recent staging systems in use. It was designed for rectum but was applied to the entire colon49. The dukes staging is as follows:
Stage A : Growth limited to the wall of rectum
Stage B : Extension of growth to extrarectal tissues, no metastasis to regional lymph nodes.
Stage C : C1-metastases to regional lymph nodes.
C2- metastases to lymph nodes at point of mesenteric blood vessel ligature
Stage D : Distant metastases.
33 FIGURE 3: DUKES STAGING OF COLORECTAL CARCINOMA
.This picture shows the extension of disease in each stage.
ASTLER- COLLER CLASSIFICATION OF STAGING OF
COLORECTAL CARCINOMA50:
Stage A : lesion limited to mucosa
Stage B : B1- lesion involves muscularis propria but doesnot penetrate through it
B2- lesion penetrates through the muscularis propria
Stage C : C1- metastatic tumour in lymph nodes but the tumour itself is still confined to the bowel wall
C2 - metastatic tumour in lymph nodes and tumour itself has penetrated through the entire bowel wall.
34 TNM Staging51
Currently TNM staging has replaced Dukes and Astler-Coller staging methods. The following is the 8th edition of American Joint cancer committee classification of TNM staging for colorectal carcinomas.
Primary tumour (pT)
Tx-Primary tumour cannot be assessed
T0-No evidence of primary tumour
Tis- Carcinoma in situ, intramucosal carcinoma
T1- tumour invades the submucosa (Through the muscularis mucosa but not into muscularis propria)
T2- tumor invades the muscularis propria
T3-tumour invades through the muscularis propria into pericolorectal issues.
T4: tumour invades the visceral peritoneum or invades or adheres to adjacent organ or structure
T4a: tumour invades through the visceral peritoneum
T4b: tumour directly invades or adheres to adjacent organs or structures.
35 Regional lymph Nodes (pN)
Nx- Regional lymph nodes cannot be assessed
N0- No regional lymph node metastasis
N1a: One regional lymph node is positive
N1b: Two or three regional lyumph nodes are positive
N1c: No regional lymph nodes are positive, but there are tumour deposits in the subserosa, mesentery or non peritonealised pericolic, or perirectal/mesorectal tissues
N2 : Four or more lymph nodes are positive
N2a : Four to six regional lyruph nodes are positive
N2b : Seven or more regional lymph nodes are positive
Distant Metastasis (pM) [Required only if confirmed pathologically]
M0 – No distant metastasis
M1 a – Metastasis to one site or organ is identified without peritoneal metastasis
M1b - Metastasis to two or more sites or organs is identified without peritoneal metastasis
36 M1c - Metastasis to the peritoneal surface is identified alone or with other site or organ metastases
FIGURE 4: TNM STAGING OF COLORECTAL CARCINOMA.
37 Stage Grouping:
Stage 0 Tis N0 M0
Stage I T1 - T2 N0 M0
Stage II A T3 N0 M0
Stage II B T4a N0 M0
Stage IIC T4b N0 M0
Stage IIIA T1 - T2 N1/N1c M0
T1 N2a M0
Stage III B T3- T4a N1/N1c M0
T2-T3 N2a M0
T1-T2 N2b M0
Stage IV A any T any N M1a
Stage IV B any T any N M1b
Stage IV C any T any N M1c
38 SURVIVAL RATE ACCORDING TO STAGE54
Colon cancer survival rates, by stage
The numbers below come from the National Cancer Institute’s
SEER database, looking at patients diagnosed with colon cancer between
2004 and 2010.
The 5-year relative survival rate for patients with stage I colon
cancer is about 92%.
For patients with stage II A colon cancer, the 5-year relative survival
rate is about 87%. For stage II B cancer, the survival rate is about
65%.
The 5-year relative survival rate for stage III A colon cancers is
about 90%. For stage III B cancers the survival rate is about 72%,
and for stage III C cancers the survival rate is about 53%.
Metastatic, or stage IV colon cancers, have a 5-year relative survival
rate of about 12%.
39 Rectal cancer survival rates, by stage
The numbers below come from the National Cancer Institute’s
SEER database, looking at patients diagnosed with rectal cancer between
2004 and 2010.
The 5-year relative survival rate for patients with stage I rectal
cancer is about 88%.
For patients with stage II A rectal cancer, the 5-year relative survival
rate is about 81%. For stage II B cancer, the survival rate is about
50%.
The 5-year relative survival rate for stage III A rectal cancers is
about 83%. For stage III B cancers the survival rate is about 72%,
and for stage III C cancers the survival rate is about 58%.
Metastatic, or stage IV rectal cancers, have a 5-year relative survival
rate of about 13%.
PROGNOSTIC FACTORS IN COLORECTAL CARCINOMA:
The College of American Pathologists have categorized the prognostic factors in colorectal carcinoma into five catagories55 as follows:
40 Category I
Well supported by the literature, generally used in patient
management and of sufficient importance to modify TNM stage groups
Category IIA
Extensively studied biologically and/or clinically. Prognostic value
for therapy, sufficient to be noted in pathology report
Category IIB
Well studied but not sufficiently established for Category I or IIA
Category III
Not yet established to meet criteria for Category I or II
Category IV
Studied and shows no consistent prognostic significance
The prognosis and the survival rate in the colorectal carcinoma depends on various factors such as age, sex ,tumor location , tumor multiplicity , local extent, tumor size, tumor edge , presence of obstruction and perforation, tumor margins and inflammatory reaction, tumor budding, vascular invasion, pericolonic tumor deposits, perineural invasion, surgical margins , tumor type, tumor grade , lymph node involvement and tumor tissue molecular markers.
41 1. AGE
Patients with very young and very old age have a poor prognosis. This may be due to the delay in diagnosis leading to more advanced stages, lesion arising in the background of ulcerative colitis and/or the presence of signet ring and mucinous tumors57.
2. SEX
The prognosis is considered to be better for females than for males56.
3. SERUM CEA LEVELS
Serum CEA levels >5.0 ng/mL have been associated with an adverse prognosis that is independent of tumor stage 55. (AJC category I).
4. TUMOR LOCATION
Lesions located in the left colon usually have a favorable prognosis, whereas those situated in the sigmoid colon and rectum have a worst prognosis. However the left-sided lesions are prone for late recurrences56.
5. TUMOR MULTIPLICITY
The survival rate for patients with synchronous or metachronous carcinomas remains the same as that of patients with solitary colorectal carcinoma56.
42 6. LOCAL EXTENT
Focal microscopic carcinoma found incidentally in a polyp and the carcinoma restricted to the mucosa and submucosa are generally associated with a good prognosis . Carcinomas that have extended beyond the bowel wall and that have metastasized to regional lymph nodes usually have a worst prognosis55.
7. TUMOR SIZE
Although a correlation between size of the tumor and prognosis exists, tumor size is not considered as a reliable prognostic indicator55.
(AJC category III).
8. TUMOR EDGE
Advanced colorectal carcinomas with a nonpolypoid edge have the worst prognosis than polypoid tumors55 (AJC category III).
9. INTESTINAL OBSTRUCTION AND PERFORATION
Patients presenting with intestinal obstruction and perforation have a worsened prognosis independent of the stage56.
10. TUMOR MARGINS AND INFLAMMATORY REACTION
Carcinomas having pushing margins and an inflammatory infiltrate at the interphase between the tumor and the neighboring tissue (made up of plasma cells and lymphocytes and associated with degenerative changes within the tumor) have a better prognosis than those lacking these
43 features55 (AJC category IIA). Also infiltration of the tumor stroma by eosinophils and dendritic cells positive for S-100 protein are associated with an improved prognosis. However, the presence of more mast cells was found to have a overall lower survival rate.
11. TUMOR BUDDING
This term relates to the epithelial mesenchymal transformation.
Tumour budding refers to the detachment and migration of neoplastic cells into the stroma at the advancing edge of the tumour. WHO postulates that tumor budding do not influence the overall grading of colorectal cancers.
Tumour budding is commonly seen in cancers with expansile or infiltrative growth pattern. It is common in cancers which are MSS, MSI-L and Lynch syndrome that is absent in sporadic MSI-H cancers24. This has been implicated as a new prognostic indicator in the management of the patient with the endoscopically removed malignant colorectal polyp. Tumor budding has emerged as a strong and independent prognostic marker of poor outcome.
12. VASCULAR INVASION
Vascular invasion significantly increases the incidence of distant metastases and decreases the 5-year survival. This feature is of more prognostic significance when it involves extramural vessels than when it is located within the bowel wall56. (AJC category IIA).
44 13. PERICOLONIC TUMOR DEPOSITS
Presence of pericolonic tumor deposits are probably the expression of a destructive type of vascular invasion, which is associated with a poor prognosis55.
14. PERINEURIAL INVASION
Perineurial invasion is usually a sign of advanced disease and associated with a poor prognosis55. (AJC category IIA).
15. SURGICAL MARGINS
Tumor involvement of the radial margin (as defined by adventitial soft tissue margin of a nonperitonealized surface) may be the single most critical factor in predicting local recurrence in rectal carcinoma . (AJC category IIA). However the risk of local recurrence in rectal adenocarcinoma after total mesorectal resection is higher if the tumor is less than 2 mm from the circumferential margin56.
16. MICROSCOPIC TUMOR TYPE
Mucinous carcinoma, signet ring cell carcinoma have a worse prognosis than the ordinary type of adenocarcinoma, whereas medullary carcinoma is said to be associated with an improved outcome55 (AJC category IIB).
45 17. ACINAR MORPHOLOGY
A microacinar pattern of growth is associated with poor prognosis but does not represent an independent prognostic factor.
18. TUMOR ANGIOGENESIS
This factor leads to recurrence and is associated with decreased survival in colorectal carcinoma. (AJC category III).
19. LYMPH NODE INVOLVEMENT
As soon as the lymph nodes show positivity for carcinomatous deposits, the 5-year survival rate drops sharply (AJC category I, as part of the staging). The location and extent of lymph node involvement are also significant. The greater the number of lymph nodes involved, the worse the prognosis.
20 MICROSCOPIC GRADE
High grade tumours are associated with poor prognosis. (AJC category IIA). It has been suggested that the grade assigned to a tumor should be determined by the worst pattern rather than the predominant one.
21 TUMOUR TISSUE MOLECULAR MARKERS55
Loss of heterozygosity(LOH) at the chromosome 18q is associated with adverse prognosis. Patients with Stage II colorectal carcinoma having 18qLOH behave clinically as Stage III patients and
Stage II patients without 18qLOH behave as Stage I patients. K-RAS
46 mutations usually occur in patients with other poor prognostic factors such as lymphatic or hematogenous metastases. Tumours developing as a result of mutations occurring in one of the DNA Mismatch Repair genes leading to microsatellite instability are associated with a favourable prognosis.
Loss of p27 have a reduced survival rate in stage III colon cancer. Loss of Bcl-2 expression in stage II colorectal carcinoma leads to increased number of relapses. P53 mutation or allelic loss decreases the probability of apoptosis and leads to unregulated cell growth. Tumours harbouring p53 mutations are at greater risk for metastasis.
ROLE OF PROLIFERATION MARKERS IN COLORECTAL
CARCINOMA :
It has been widely accepted that the colorectal carcinomas of the same pathologic stage can produce different clinical outcomes.
Therefore determination of other factors that influence the rate of increase in tumour volume is recommended. The rate of tumour growth depends on its proliferative activity and cell death. Cell proliferation is a key feature in the progression of tumour58.
Antibodies that recognize nuclear proteins associated with tumour cell proliferation can be determined by immunohistochemistry and
47 represent an attractive alternative to the analysis of cell proliferation by flow cytometry. Many proliferation and anti-apoptotic markers such as
PCNA , P53, Ki67,AGNOR,Statin,BrdU and Bcl-2 are being studied60.
Currently, Ki-67, and its epitope MIB-1, is the most popular antibody which has been investigated as a prognostic factor in colorectal carcinoma58.
Figure 5: Schematic diagram of Ki67 as a promising molecular target in the
diagnosis of cancer. The expression of Ki-67 is associated with tumor cell
proliferation and growth. The nuclear protein Ki-67 (pKi-67) is an established
prognostic and predictive indicator for the assessment of cell proliferation in
biopsies from cancer patients.
48 Ki67
Ki-67 is a proliferation associated nuclear antigen. Ki67 is a non- histone protein expressed in all cycling cells except resting cells in the G0 phase, and it reflects cells in the S/G2+M phase, in particular. The Ki-67 gene is present on the long arm of the human chromosome 10 (10q25).The half life of Ki-67 protein has been estimated about 60-90 minutes. The Ki-
67 antigen is detected in G1, S, G2 and M phases of cell cycle but not in
G0 phase. Though the flow cytometry methods showed that Ki-67 is expressed in all the cell cycle phases except G0 phase, its detection by immunohistochemical methods is limited to its appearance in the late G1 phase with maximum expression in prophase and metaphase. Then its immunoexpression diminishes in anaphase and telophase, its levels being apparently undetectable during most of the interphase . For these reasons,
Ki-67 is recognized as a mitotic activity indicator58.A rise in Ki-67 expression indicates a rise of the mitotic activity and of the cell proliferation.
The Ki-67 protein is phosphorylated via serine and threonine with a critical role in cell division. This has been observed from the arrest of cell proliferation when Ki-67 is blocked either by microinjection of blocking antibodies or by inhibition of dephosphorylation60.
49 The Ki-67 expression is estimated as the percentage of the tumour cells positively stained by the antibody, with nuclear staining being most common criteria of positivity. MIB-1 is a monoclonal antibody and it recognizes the Ki-67 nuclear antigen in the formalin fixed paraffin embedded tissue sections and its reactivity is not affected if there is a delay in fixation59.
Expression of Ki67, as evaluated by immunostaining has become the gold standard, with a cutoff level of between 10 and 14% positively-stained cells defined as high risk in terms of prognosis 61. The St
Gallen Consensus in 2009 considered the Ki-67 labelling index as an important factor in selecting the chemotherapy in addition to endocrine therapy in hormone receptor-positive breast cancers. Tumors were classified as low, intermediate, and highly proliferating, according to the
Ki-67 labelling index when the index was ≤15%, 16%–30%, and >30%, respectively 62.
In a study done by Georgesu et al, the mean Ki-67 labelling index increased with the histological grade of colorectal adenocarcinomas and had the following values: 20% in well differentiated adenocarcinoma
(ranges 14-23%), 34% in moderately (ranges 18-57%), and 57% in poorly differentiated adenocarcinoma (ranges 35-87%) . The difference between grade 1 and grade 2 adenocarcinomas was not significant, while the
50 difference between grade 2 and 3 adenocarcinomas was significant
(p<0.05)63.
51 MATERIALS AND METHODS
STUDY DESIGN: Both retrospective and prospective analysis.
STUDY AREA: This study was conducted in the Department of
Pathology, Tirunelveli Medical college hospital.
STUDY SAMPLE: This study was conducted on the 42 cases of colorectal carcinoma reported in the of Department of Pathology,
Tirunelveli Medical College during the period 2015 to 2018.
STUDY POPULATION: This includes cases reported as colorectal cancer in the Department of Pathology, TVMC.
INCLUSION CRITERIA: Specimens received in the histopathological laboratory reported as colorectal carrinoma.
EXCLUSION CRITERIA: 1.Samples received from patients of
Colorectal carcinoma who are subjected to prior chemotherapy and /or radiotherapy. 2.Small biopsies of cases of colorectal carcinoma.
DURATION OF STUDY: 18 months (March 2017 to September 2018)
METHODOLOGY:
Paraffin blocks containing tissue of surgically resected colorectal carcinoma patients were collected from the files of the Department of
Pathology in Tirunelveli medical college. The Haematoxylin and Eosin
52 staining64 was done on the tissue sections of 4-5 µm thickness were obtained from each of the paraffin block on albumin coated slides followed by dewaxing by incubation of slides at 60-70o C for one hour. Sections were then rehydrated using graded alcohols and brought to water. Staining with Harris hematoxylin was done for 10 minutes to stain all the nuclei.
The sections were then washed for 5 minutes under running tap water for blueing, counterstained in 1% aqueous Eosin for 8 dips and then washed in tap water. The sections were dehydrated, cleared and mounted.
Sections stained with haematoxylin and eosin were used for histological typing and grading of colorectal carcinomas. The studied 42 cases of colorectal carcinoma were classified according to WHO histological classification47 as Adenocarcinoma,NOS type (34 cases), mucinous adenocarcinomas (7 cases) and squamous cell carcinoma (1 case). Adenocarcinomas are graded according to the degree of tubule formation and cellular array in tumor tissue as : Grade 1 or Well differentiated - >95% with gland formations, tumour cells had a uniform appearance and there is no or minimal loss of polarity ; Grade 2 or
Moderately differentiated - 50-95% with gland formation, tubular structures can be simple or complex as may be slightly irregular shaped , loss of nuclear polarity was evident; Grade 3 or Poorly differentiated -
0-49% with gland formation65 . The pathological tumor staging was
53 performed according to the American Joint Committee on cancer by grouping the various TNM components51. The tumor stage (T) was determined from sections that reached the pericolic fat; the nodal status (N) was also recorded from corresponding sections and any metastasis (M) was reported from the patient file and accompanying specimens.
Tumour cell proliferation was studied by doing immunohistochemistry with Ki67 antibody. Immunohistochemical staining was done in the normal intestinal mucosa of patients in the age group of 2nd to 7th decade which were subjected to histopathological examination served as the control group and 42 cases of colorectal carcinoma which was the study group. An analysis was done by correlating the Ki67 index with the stage and grade of the tumour in these 42 cases of colorectal carcinoma.
IMMUNOHISTOCHEMISTRY FOR Ki67
Special positively charged, poly-L-lysine coated slides are used for
IHC. Thin sections of about 3-4µm size was cut on the charged slides.
Steps in IHC66
1. Charged slides with 3-4µm thick section are incubated at 60-70o C for
1 hour for dewaxing.
2.Sections were deparaffinized , hydrated through descending grades of alcohol and then brought to water.
54 3.Antigen Retrieval was done in Pressure Cooker by keeping the slides in a pre-heated Tris-EDTA retrieval buffer of pH -9 at 60o C for 20 minutes.The dilution used for Tris – EDTA retrieval buffer was 1 in 50.
Then the slides were washed with distilled water for two minutes.
4. Slides were treated with Tris-Wash buffer of pH 7.6 at room temperature for 5 minutes. The dilution of Tris-Wash Buffer was 1 in 20.
5. Slides were treated with peroxidase blocking reagent for 15 minutes.
6. Then washed in Tris-Wash buffer for 5 minutes.
7. Slides were treated with Ki-67 primary antibody for 30 minutes.
8. Then washed in Tris -Wash buffer for 5 minutes.
9.Slides were treated with HRP Polymerase, secondary antibody for 30 minutes.
10. Then washed in Tris-Wash buffer for 5 minutes.
11. Slides are covered with substrate buffer containing DAB
(Diaminobenzidine tetrachloride) chromogen for 10 minutes.
12. Then washed in Tris-Wash Buffer for 5 minutes.
13. Slides were then washed with Distilled water two changes.
55 14. Counterstaining was done with Mayer’s Haematoxylin for 30 seconds to impart background staining.
15. Running tap water wash given.
16. Xylene, 2 changes 5 minutes each was done.
17. Dehydration in 100% alcohol for 5 minutes.
18. Then the sections were mounted with DPX. (Dextrene Phthalate
Xylene)
CALCULATION OF Ki67 INDEX IN THE NORMAL INTESTINAL
MUCOSA67
Ki67 index in the normal intestinal mucosa was calculated by counting the positively stained (labeled) cells in at least five full longitudinal crypts. For each crypt, the number of cells per crypt column and the number of labeled cells along the crypt were counted and recorded. Each crypt was divided into three equal compartments, upper(surface), middle and lower. The labeling index was calculated for the entire crypt . The whole – crypt labeling index was the percentage of the total number of the labeled cells in each crypt.
x100
Ki67 whole crypt labelling index =
56 Ki67 Labelling Index in colorectal carcinoma cases:
Ki 67 scoring for the 42 colorectal carcinoma cases was done by counting atleast 1000 tumor cells in atleast 4-5 high power fields. Every stained nucleus was considered positive, irrespective of intensity. The percentage of positively stained cells was recorded as Ki67- labeling index68,77.
x100
Ki67 labelling index = Statistical analysis
Data were entered in Microsoft Excel and statistical analysis was done using the software SPSS 16.0.version .Descriptive statistics like
Mean ± Standard deviation was done for quantitative variables and proportion or percentage for categorical variables.
57 OBSERVATION AND RESULTS
A total of 42 formalin fixed colectomy/ hemicolectomy specimens from patients with colorectal carcinoma were included in this study.
Among these patients, 20 cases were males and 22 cases were females.
The minimum age of presentation was 17 years and the maximum age of presentation was 76 years. The mean age of presentation was 53.95 years.
DISTRIBUTION OF CASES ACCORDING TO SITE
In the 42 cases, 8 cases were found in the Ascending colon (19%), 6 cases in the Caecum(14.3%), 3 cases in the Descending colon(7.1%),4 cases in the Transverse colon(9.5%), 3 cases in the Recto- sigmoid junction(7.1%), 10 cases in the Rectum(23.8%) , and 8 cases in the Sigmoid colon(19%). [Table 1 & Chart 1].
TABLE 1: DISTRIBUTION OF CASES ACCORDING TO SITE
Site Number of cases Percentage
ASCENDING COLON 8 19.0
CAECUM 6 14.3
DESCENDING COLON 3 7.1
RECTO-SIGMOID JUNCTION 3 7.1
RECTUM 10 23.8
SIGMOID COLON 8 19.0
TRANSVERSE COLON 4 9.5
Total 42 100.0
58 CHART 1: DISTRIBUTION OF CASES ACCORDING TO SITE
SITE
12% 23% ASCENDING COLON CAECUM DESCENDING COLON 29% RECTO-SIGMOID JUNCTION
18% RECTUM TRANSVERSE COLON
9% 9%
DISTRIBUTION OF CASES ACCORDING TO HISTOLOGICAL TUMOUR
TYPE
Out of the 42 cases, 34 cases were found to be
Adenocarcinoma, NOS type(81%), 7 cases were Mucinous adenocarcinoma(16.7%) and 1 case was Squamous cell carcinoma(2.4%).[Table 2 & Chart 2].
59 TABLE 2: DISTRIBUTION OF CASES ACCORDING TO HISTOLOGICAL
TUMOR TYPE
Histological Type Number of Percentage
cases
Adenocarcinoma 34 81.0
Mucinous adenocarcinoma 7 16.7
Squamous cell carcinoma 1 2.4
Total 42 100.0
CHART 2: DISTRIBUTION OF CASES ACCORDING TO HISTOLOGICAL
TUMOUR TYPE
HISTOLOGICAL TUMOR TYPE
2%
17%
ADENOCARCINOMA MUCINOUS ADENOCARCINOMA SQUAMOUS CELL CARCINOMA
81%
60 DISTRIBUTION OF ADENOCARCINOMA CASES ACCORDING TO GRADE
Among the 34 cases of adenocarcinoma, 14 cases were well- differentiated(41.2%), 16 cases were moderately-differentiated(47%) and 4 cases were poorly-differentiated(11.8%).[Table 3 & Chart 3].
TABLE 3: DISTRIBUTION OF ADENOCARCINOMA CASES ACCORDING
TO GRADE
Grade Number of cases Percentage WELL DIFFERENTIATED 14 41.2 MODERATELY DIFFERENTIATED 16 47.0 POORLY DIFFERENTIATED 4 11.8 Total 34 100.0
CHART 3: DISTRIBUTION OF ADENOCARCINOMA CASES ACCORDING
TO GRADE
GRADES
2%
46% WELL DIFFERENTIATED 52% MOD. DIFFERENTIATED POORLY DIFFERENTIATED
61 DISTRIBUTION OF CASES ACCORDING TO STAGE
In the 42 cases, 3 cases were in stage I (7.1%) , 11 cases in stage
IIA (26.2%) , 6 cases in stage II B(14.3%) ,5 cases in stage II C(11.9%) ,
1 case in stage III A(2.4%) , 10 cases in stage III B(23.8%) , 5 cases in stage III C(11.9%) , 1 cases in stage IV C(2.4%) . [Table 4 & Chart 4].
TABLE 4: DISTRIBUTION OF CASES ACCORDING TO STAGE
Stage Number of cases Percentage
I 3 7.1
II A 11 26.2
II B 6 14.3
II C 5 11.9
III A 1 2.4
III B 10 23.8
III C 5 11.9
IV C 1 2.4
Total 42 100.0
62 CHART 4: DISTRIBUTION OF CASES ACCORDING TO STAGE
STAGES
2%
7% 12% I II A
26% II B II C 24% III A III B III C IV C 14% 3% 12%
63 CORRELATION OF THE STAGE, GRADE AND Ki67 INDEX
The Ki-67 index of the normal intestinal mucosa of patients in the age group of 2nd to 7th decade which were subjected to histopathological examination ranged from 2.6% to 18.2 % with a mean Ki-67 index of
9.992% . [Table 5,Figure(i)]
TABLE:5 Ki67 INDEX IN THE NORMAL INTESTINAL MUCOSA
Number Minimum Maximum Mean Std. Deviation of cases value value
Ki67 INDEX 15 2.6 18.2 9.992 4.910
64 Positive nuclear immunohistochemical staining for Ki-67 antibody was seen in all 42 cases of colorectal carcinomas. Ki67 index was calculated for all the 42 cases.The minimum Ki67 index observed was 14.5% and the maximum index obtained was 74.6%.[Table 6 & Chart 5].
TABLE:6 UPPER AND LOWER VALUES OF i67 INDEX IN THE STUDY
Number Minimum Maximum Mean Std. Deviation
of cases value value
Ki67 INDEX 42 14.5 74.6 50.498 15.8879
CHART 5: UPPER AND LOWER VALUES OF Ki67 INDEX IN THE STUDY
UPPER AND LOWER VALUES OF ki67 INDEX IN THE STUDY
Ki67_INDEX, 74.6 80 70 60 50 40 30 Ki67_INDEX, 14.5 20 10 0 Ki67_INDEX
Minimum Maximum
65 The mean Ki67 index for the 14 cases of well-differentiated
adenocarcinomas was 54.157 , 16 cases of moderately differentiated
carcinomas was 50.838, for 4 cases of poorly differentiated carcinomas
was 63.950, 7 cases of mucinous adenocarcinomas was 33.343 and 1 case
of squamous cell carcinoma was 60.10. Mucinous adenocarcinomas
showed the low proliferating index . [Table 7,Figure(ii) to (ix)].
TABLSE 7 : STATISTICAL ANALYSIS OF Ki67 INDEX
95% Confidence Std. Std. Interval for N Mean Minimum Maximum Deviation Error Mean Lower Upper Bound Bound
WELL 14 54.157 11.7995 3.1536 47.344 60.970 36.5 66.8 DIFFERENTIATED
MODERATELY 16 50.838 15.5313 3.8828 42.561 59.114 24.3 74.6 DIFFERENTIATED
POORLY 4 63.950 6.6496 3.3248 53.369 74.531 59.1 73.4 DIFFERENTIATED
MUCINOUS 7 33.343 16.9463 6.4051 17.670 49.016 14.5 67.5 ADENOCARCINOMA
SQUAMOUS CELL 1 60.100 . . . . 60.1 60.1 CARCINOMA
66 The mean Ki67 index for the 3 cases of stage I was 61.80, 11 cases of stage II A was 47.10, 6 cases in stage II B was 47.76, 5 cases in stage
II C was 49.32, 1 case in stage III A was 34.80, 10 cases in stage III B was
54.36, 5 cases in stage III C was 51.00, 1 case in stage IV C was 50.80.
The highest proliferative index was observed in stage I. [Table 8].
TABLE 8 : STATISTICAL ANALYSIS OF Ki67 INDEX IN DIFFERENT
STAGES
95% Confidence Std. Std. Interval for N Mean Minimum Maximum Deviation Error Mean Lower Upper Bound Bound STAGE 3 61.800 2.2650 1.3077 56.174 67.426 59.4 63.9 I
II A 11 47.100 12.0864 3.6442 38.980 55.220 30.3 65.8
II B 6 47.767 16.8435 6.8763 30.091 65.443 24.3 66.8
II C 5 49.320 12.0157 5.3736 34.401 64.239 35.0 60.1
III A 1 34.800 . . . . 34.8 34.8
III B 10 54.360 16.8324 5.3229 42.319 66.401 23.7 71.4
III C 5 51.000 29.0411 12.9876 14.941 87.059 14.5 74.6
IV C 1 50.800 . . . . 50.8 50.8
Total 42 50.498 15.8879 2.4516 45.547 55.449 14.5 74.6
67 ANALYSIS OF Ki67 INDEX OF EACH CASE UNDER STUDY
In this study, out of 42 cases , 3 cases were in stage I. Among the
3 cases, 1 case belongs to grade 1 which has a Ki67 index of 63.9 and 2 cases belong to grade 2 which have the mean Ki67 index of 60.75 [Table
9 & Chart 6].
TABLE 9: CASES OF STAGE I WITH GRADE AND Ki67 INDEX
Stage Grade No. of Cases Ki 67 index of Mean ki67 of each case each grade
Grade 1 1 63.9 63.9
Stage I Grade 2 2 59.4 60.75 62.1
Total 3
CHART 6: CASES IN STAGE I WITH GRADE AND MEAN Ki67 INDEX
STAGE I WITH GRADE AND Ki67 INDEX
63.9
64
63
62 60.75
61
60
59 Stage I
Grade 1 Grade 2
68 Out of 42 cases, 11 cases were in stage II A. Among the 11 cases, 6 cases belongs to grade 1 which had the mean Ki67 index of 48.683 , 4 cases belong to grade 2 which had the mean Ki67 index of 48.925 and 1 case was the mucinous adenocarcinoma which has a Ki67 index of 30.3[Table
10 & Chart 7].
TABLE 10: CASES OF STAGE II A WITH GRADE AND Ki67 INDEX
Ki67 index Mean Ki67 of Stage Grade No. of Cases of each case each grade Grade 1 6 38 48.683 38.9 40.2 47.8 63.3 64.2 Grade 2 4 38.1 48.925 Stage II A 45 46.8 65.8 Mucinous 1 30.3 30.3 Total 11 CHART 7: CASES IN STAGE II A WITH GRADE AND MEAN Ki67 INDEX
STAGE II A WITH GRADE AND Ki67 INDEX
48.683 48.925
50 45 40 30.3 35 30 25 20 15 10 5 0 Stage II A
Grade 1 Grade 2 Mucinous
69 Out of 42 cases, 6 cases were in stage II B. Among the 6 cases, 3 cases belongs to grade 1 which had the mean Ki67 index of 56.30 and 3 cases belong to grade 2 which had the mean Ki67 index of 39.23. [Table
11 & Chart 8].
TABLE 11: CASES OF STAGE II B WITH GRADE AND Ki67 INDEX
Ki67 index Mean Ki67 of Stage Grade No. of Cases of each case each grade Grade 1 3 36.5 56.30 65.6 66.8 Grade 2 3 24.3 39.23 Stage II B 41.3 52.1 Total 6 CHART 8: CASES IN STAGE II B WITH GRADE AND MEAN Ki67 INDEX
STAGE II B WITH GRADE AND Ki67 INDEX
60 50 40 30 20 10 0 Stage II B
Grade 1 Grade 2
70 Out of 42 cases, 5 cases were in stage II C. Among the 5 cases, 1 case belongs to grade 1 which had the Ki67 index of 54.6 , 1 case belong to grade 3 which had the Ki67 index of 59.1 and 2 cases were the mucinous adenocarcinoma which had the mean Ki67 index of 36.4, 1 case was a squamous cell carcinoma which had the Ki67 index of 60.1 . [Table 12 &
Chart 9].
TABLE 12: CASES OF STAGE II C WITH GRADE AND Ki67 INDEX
Ki67 index Mean Ki67 of Stage Grade No. of Cases of each case each grade Grade 1 1 54.6 54.6 Stage II C Grade 3 1 59.1 59.1 Mucinous 2 35 36.4 37.8
Squamous cell 1 60.1 60.1 carcinoma
Total 5
CHART 9: CASES IN STAGE II C WITH GRADE AND MEAN Ki67 INDEX
STAGE II C WITH GRADE AND Ki67 INDEX
59.1 60.1 70 54.6 60 50 36.4 40 30 20 10 0 Stage II C
Grade 1 Grade 3 Mucinous SQUAMOUS CELL CARCINOMA
71 Out of 42 cases, 1 case in stage III A which was grade 1 adenocarcinoma had the Ki67 index of 34.8 .[Table 13 & Chart 10].
TABLE 13: CASES OF STAGE III A WITH GRADE AND Ki67 INDEX
Ki 67 index of Stage Grade No. of Cases each case
Grade 2 1 34.8
Stage III A Total 1
CHART 10: CASES IN STAGE III A WITH GRADE AND MEAN Ki67 INDEX
STAGE III A WITH GRADE AND Ki67 INDEX
34.8
35 30 25 20 15 10 5 0 Stage III A
Grade 2
72 Out of 42 cases, 10 cases were in stage III B. Among the 10 cases, 2 cases belongs to grade 1 which had the mean Ki67 index of 63.95, 4 cases belongs to grade 2 which had the mean Ki67 index of 50.30, 2 cases belong to grade 3 which had the mean Ki67 index of 61.65 and 2 cases were the mucinous adenocarcinoma which had the mean Ki67 index of
45.60. [Table 14 & Chart 11].
TABLE 14: CASES OF STAGE III B WITH GRADE AND Ki67 INDEX
Ki67 index Mean Ki67 of Stage Grade No. of Cases of each case each grade Stage III B Grade 1 2 63.2 63.95 64.7 Grade 2 4 25.5 50.30 50.8 53.5 71.4 Grade 3 2 59.5 61.65 63.8 Mucinous 2 23.7 45.60 67.5 Total 10 CHART 11: CASES IN STAGE III B WITH GRADE AND MEAN Ki67 INDEX
STAGE III B WITH GRADE AND Ki67 INDEX
63.95 61.65 70 50.3 60 45.6 50 40 30 20 10 0 Stage III B
Grade 1 Grade 2 Grade 3 MUCINOUS
73 Out of 42 cases, 5 cases were in stage III C. Among the 5 cases,
2 cases belongs to grade 2 which had the mean Ki67 index of 71.25, 1 case belong to grade 3 which had the Ki67 index of 73.4 and 2 cases were the mucinous adenocarcinoma which had the mean Ki67 index of
19[Table 15 & Chart 12].
TABLE 15: CASES OF STAGE III C WITH GRADE AND Ki67 INDEX
Mean Ki67 Ki67 index of Stage Grade No. of Cases of each each case grade
Stage III C Grade 2 2 67.9 71.25 74.6
Grade 3 1 73.4 73.4
MUCINOUS 2 14.5 19.55 24.6
Total 5
CHART 12: CASES IN STAGE III C WITH GRADE AND MEAN Ki67 INDEX
STAGE III C WITH GRADE AND Ki67 INDEX
71.25 73.4 80 70 60 50 40 19.55 30 20 10 0 Stage III C
Grade 2 Grade 3 MUCINOUS
74 Out of 42 cases in the study, 1 case in stage IV C which was grade 1 adenocarcinoma had the Ki67 index of 50.8. [Table 16 & Chart
13].
TABLE 16: CASES OF STAGE IV C WITH GRADE AND Ki67 INDEX
Ki67 index of Stage Grade No. of Cases each case
Stage IV C Grade 1 1 50.8
Total 1
CHART 13: CASES IN STAGE IV C WITH GRADE AND MEAN Ki67 INDEX
STAGE IV C WITH GRADE AND Ki67 INDEX
Grade 1
50.8
Stage IV C
75 In this study of 42 cases of colorectal carcinomas , the mean Ki67 index for the well-differentiated ( grade 1) adenocarcinomas was 54.15 ±
11.79%,moderately differentiated ( grade 2) adenocarcinomas was
50.83±15.53%, for poorly differentiated ( grade 3) adenocarcinomas was
63.95 ± 6.64% and for mucinous adenocarcinomas was 33.343 ± 16.94% .
The difference between grade 1 and grade 2 adenocarcinomas was not significant while the difference between grade 1 and grade 3 adenocarcinomas was significant and the difference between grade 2 and grade 3 adenocarcinomas was significant .
The lowest Ki67 index of 14.5 was observed in a case of mucinous adenocarcinoma and the highest ki67 index of 73.4 was observed in the case of grade 3 carcinoma in stage III C. Thus in these cases, the stage and grade of tumor correlates well with the proliferating potential of the tumour.
In our study of 42 cases, the 3 cases in stage I had a high proliferative index though they have a low grade while comparing the mean value. Out of 11 cases in stage II A, 3 cases had a high proliferative index inspite of having a low grade. And also 2 cases out of 6 cases in stage II B had a high proliferative index inspite of having a low grade and being in low stage. We also a observed low Ki67 index of 34.8% in a case
76 of grade 2 carcinoma of stage III A and a low Ki67 index of 25.5% in a case of grade 2 carcinoma of stage III B.
Out of the 7 cases of mucinous adenocarcinoma , 6 cases(85.7%) have the Ki67 index of 14.5, 23.7 , 30.3, 33.5,35 and 37.5 , which was observed to be low. But a high Ki67 index of 67.5 was observed in a stage
III B mucinous adenocarcinoma.
Therefore, 8 cases in the study had a high proliferative index inspite of having a low grade and stage, 2 cases had a low proliferative index inspite of being in the higher stage and 1 case of mucinous adenocarcinoma had a high proliferative index . These 11 cases (26.2 %) could show a different biological behavior when comparing with other cases in the same stage and grade at presentation. However, stage I tumors could have a high proliferative index. Hence the other 5 cases with high proliferative index in the lower stage needs to be followed up carefully for recurrence and distant metastasis and the 2 cases with low proliferative index in the higher stage can have a better survival. [Table 17] .
77 TABLE 17: CASES WITH Ki67 INDEX NOT CORRELATING TO STAGE
AND GRADE OF PRESENTATION
No. STAGE GRADE Ki67 INDEX
1. Stage II A Grade 1 63.3 2. Stage II A Grade 1 64.2 3. Stage II A Grade 2 65.8 4. Stage II B Grade 1 65.6 5. Stage II B Grade 1 66.8 6. Stage III A Grade 2 34.8 7. Stage III B Grade 2 25.5
8. Stage III B Mucinous 67.5 Adenocarcinoma
Out of 42 cases, 8 cases (19%) had a Ki67 proliferation index that didnot correlate with their stage and grade at presentation. Thus 19% of the cases showed a different biological behavior from the other cases in the same stage and grade were picked up with the help of Ki67 proliferation index.
While taking Ki67 proliferation index along with histological grade and stage of the tumour into account we were able to provide an additional tool in deciding the prognosis and further management of the patient.
78 DISCUSSION
Colorectal cancer was the most common cause of cancer death in the late 1940s and early 1950s. Today, it is the third leading cause of cancer death in both men and women, as a consequence of changes in lifestyle risk factors (e.g., decreased smoking and red meat consumption and increased use of aspirin), the introduction and distribution of early detection tests, and improvements in treatment.
The 5-year survival rate is high for stage I and II A patients and decreases down the stage69. Tumours with grade 1 and 2 differentiation are associated with a better prognosis compared to grade 3 tumors. The prognosis of mucinous adenocarcinoma in comparison with conventional adenocarcinoma has been controversial among different studies. Many mucinous adenocarcinomas occurring in patients with hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) represent highlevel MSI (MSI-H) tumors. These tumors are expected to behave in a low grade fashion. In contrast, mucinous adenocarcinomas that are microsatellite stable (MSS) are expected to behave more aggressively, particularly when detected at an advanced stage70.Also the mucinous adenocarcinomas have a poorer response to chemotherapy on account of its high mucin content and low cellularity71.
79 Cellular proliferation is essential to maintain tissue homeostasis and is imperative in oncogenesis. Assessment of tumour cell proliferation may predict tumour behaviour. Quantification of cell proliferative activity in neoplasia is currently the subject of considerable investigation. The Ki-
67 is a nuclear antigen present during all active phases of the cell cycle
(G1, S, G2 and M), but is absent in resting cells (G0). In later phases of mitosis (during anaphase and telophase), a sharp decrease in Ki67 levels occurs. Expression of the Ki67 protein (pKi67) is associated with the proliferative activity of intrinsic cell populations in malignant tumors, allowing it to be used as a marker of tumor aggressiveness58,59,68. The prognostic value of pKi67 has been investigated in a number of studies with its potential as a reliable marker having been shown in cancers of the breast, gastrointestinal tract, soft tissue, lung, prostate, cervix and central nervous system . It is therefore widely used as a cell proliferation marker to grade tumours79.
In this study, we studied 42 cases of colorectal carcinoma in
Southern Tamilnadu. Similar studies were done by Georgescu et al with
41 cases of colorectal carcinoma admitted to the Emergency County
Clinical Hospital of Craiova in 200563, Bhagyalakshmi et al with 51 patients of CRC in the Andhra Medical College, Vishakapatnam during the period August 2011-July 201372 , Anway Sen et al with 74 total cases of colorectal cancers in Institute of Post-Graduate Medical Education and
80 Research (IPGMER), Kolkata, West Bengal, India during July 2010 to
June 201273 and W. LI et al with 200 cases of colorectal cancer from
January 2011 to January 2016 in Cangzhou Central Hospital, China74.
In our study of 42 cases, 8 cases were found in the Ascending colon
(19%), 6 cases in the Caecum (14.3%), 3 cases in the Descending colon
(7.1%),4 cases in the Transverse colon(9.5%), 3 cases in the Recto- sigmoid junction(7.1%), 10 cases in the Rectum(23.8%) , and 8 cases in the Sigmoid colon(19%). Rectum was the commonest site of occurrence of colorectal carcinoma. Phipps et al in their study of 3284 cases of colorectal carcinoma found that 24% cases were located in rectum and rectum as the commonest site of occurrence of colorectal carcinoma75 . This observation correlates well with our study.
Out of the 42 cases in our study, 34 cases were found to be
Adenocarcinoma, NOS type(81%), 7 cases were Mucinous adenocarcinoma(16.7%) and 1 case was Squamous cell carcinoma(2.4%).Most of the colorectal carcinomas were adenocarcinomas. This correlated well with the study conducted by
Fleming et al48 which stated that more than 90% of the colorectal carcinomas were adenocarcinomas.
Among the 34 cases of adenocarcinoma, 14 cases were well- differentiated(41.2%), 16 cases were moderately-differentiated(47%) and 4 cases were poorly-differentiated(11.8%). Moderately differentiated
81 adenocarcinoma was the most common histopathological grade observed.
Anway sen et al also observed moderately differentiated adenocarcinoma as the most common histopathological grade in their study which was similar to our study73.
In the 42 cases, 3 cases were in stage I (7.1%) , 11 cases were in stage II A (26.2%) , 6 cases were in stage II B(14.3%) ,5 cases were in stage II C(11.9%) , 1 case in stage III A(2.4%) , 10 cases were in stage III
B(23.8%) , 5 cases were in stage III C(11.9%) and 1 case in stage IV
C(2.4%) . The maximum number of cases at presentation were in stage II in our study. Li et al observed maximum number of cases in stage III at presentation74.
In our study the mean Ki-67 index of 9.99% was observed in the cases with normal intestinal mucosa with the values ranged from
2.6% to 18.2 % . Berenzi A et al observed the Ki-67 index in normal intestinal mucosa from 1.5% to 17 %76. Ki67 index was calculated for the
42 cases of colorectal carcinomas ranges from 14.5% to 74.6% with a mean Ki67 index of 50.49 %. Bhaghyalakshmi et al assessed the Ki-67 proliferation index in colorectal carcinoma which ranged from 8.4% to
84.4%72. There is a significant difference in the mean Ki-67 index of the normal intestinal mucosa and colorectal carcinoma cases which is about 5 times the normal intestinal mucosa index.
82 Regarding the histological grade of colorectal adenocarcinomas,the tumor proliferative activity detected with Ki-67 antibody increased with the decrease of the cell differentiation. The stepwise increase of mean Ki-67 index with the dedifferentiation of the colorectal adenocarcinomas indicates a cell hyperproliferation in poorly differentiated adenocarcinomas.
Anway Sen et al observed the mean Ki-67 index of well differentiated, moderately differentiated and poorly differentiated adenocarcinomas were 14.25, 31.34 and 43.08 respectively73.This difference was statistically significant. In a study done by Claudia
Valentina Georgescu et al also the mean Ki-67 index increased with the histological grade of adenocarcinomas and had the following values: 20% in well differentiated adenocarcinoma (ranges 14-23%), 34% in moderately (ranges 18-57%), and 57% in poorly differentiated adenocarcinoma (ranges 35-87%) .The difference between grade 1 and grade 2 adenocarcinomas was not significant,while the difference between grade 2 and 3 adenocarcinomas was significant (p<0.05)63. These findings correlated with our present study of 42 cases of colorectal carcinomas where the mean ki67 index for the well-differentiated ( grade 1) adenocarcinomas was 54.15 ± 11.79%,moderately differentiated ( grade 2) adenocarcinomas was 50.83±15.53%, for poorly differentiated ( grade 3)
83 adenocarcinomas was 63.95 ± 6.64% and for mucinous adenocarcinomas was 33.343 ± 16.94% . The difference between grade 1 and grade 2 adenocarcinomas was not significant while the difference between grade 1 and grade 3 adenocarcinomas was significant and the difference between grade 2 and grade 3 adenocarcinomas was significant .
In the present study, the lowest ki67 index of 14.5% was observed in a case of mucinous adenocarcinoma and the highest ki67 index of 73.4% was observed in the case of grade 3 carcinoma in stage III C .
Out of 42 cases in the study, 8 cases had a high proliferative index inspite of having a low grade and stage, 2 cases had a low proliferative index inspite of being in the higher stage and 1 case of mucinous adenocarcinoma had a high proliferation index . Georgesu et al in their study observed a high proliferation index of 55% in cases of mucinous adenocarcinoma63. High tumour proliferative index in mucinous adenocarcinoma if associated with microsatellite stability had a better response to antineoplastic chemotherapeutic agents and radiation therapy43.
Kubota et al in their study observed that stage I tumours had a high proliferation index78. In our study also, 3 cases in stage I had a high proliferation index. Hence the other 5 cases with high proliferation index in the lower stage needs to be followed up carefully for recurrence and
84 distant metastasis and the 2 cases with low proliferative index in the higher stage could have a better survival.
Among the 42 cases in our study, 8 cases (19%) had a Ki67 proliferation index that didn’t correlate with their stage and grade at presentation. These 8 cases (19%) could show a different biological behavior from the other cases in the same stage and grade in terms of their
Ki67 proliferation index. Thus by calculating the Ki67 proliferation index for cases of colorectal carcinoma , in addition to the pathological staging and grading, we were able to add a valuable supplementary prognostic indicator regarding the biological behavior of the tumour. Hence Ki67 proliferation index along with histological grade and stage of the tumour could be an additional tool in deciding the prognosis and further management of the patient.
85 SUMMARY AND CONCLUSION
This study was conducted in the Department of Pathology,
Tirunelveli Medical College hospital on the 42 cases of colorectal carcinoma reported in our department. The grading and staging of these 42 cases of colorectal carcinoma were assessed in the haematoxylin and eosin stained sections of the tumour tissue, followed by immunohistochemical analysis using Ki67 antibody was performed for assessing the proliferative indices of these 42 cases.
Among the 42 cases, 34 cases were found to be Adenocarcinoma,
NOS type(81%), 7 cases were Mucinous adenocarcinoma(16.7%) and 1 case was Squamous cell carcinoma(2.4%). Of the 34 cases of adenocarcinoma, 14 cases belong to well-differentiated or grade 1(41.2%),
16 cases belong to moderately-differentiated or grade 2 (47%) and 4 cases belong to poorly-differentiated or grade 3 (11.8%).
While staging the 42 cases, 3 cases came under stage I
(7.1%) , 11 cases came under stage II A (26.2%) , 6 cases came under stage II B(14.3%) , 5 cases came under stage II C(11.9%) , 1 case came under stage III A(2.4%) , 10 cases came under stage III B(23.8%) , 5 cases came under stage III C(11.9%) , 1 cases came under stage IV
C(2.4%) .
86 All 42 cases of colorectal carcinomas showed positive nuclear immunohistochemical staining for the proliferative marker , Ki-67. The
Ki67 index calculated for the 42 cases of colorectal carcinomas ranged from 14.5% to 74.6%. The Ki67 index for the normal intestinal mucosa index ranged from 2.6% to 18.2 % with a mean Ki-67 index of 9.99%.
Thus a significant increase in Ki67 index was observed in the cases of colorectal carcinoma.
The mean Ki67 index for the 14 cases of well-differentiated adenocarcinomas was 54.15% , 16 cases of moderately differentiated carcinomas was 50.83%, for 4 cases of poorly differentiated carcinomas was 63.95%, 7 cases of mucinous adenocarcinomas was 33.34% and 1 case of squamous cell carcinoma was 60.1%.Thus the Ki-67 index increased with the histological grade of colorectal adenocarcinomas and the mucinous adenocarcinomas were having a low proliferating index .
However not all cases in the study showed direct correlation tumour grade,stage and Ki67 index. Among the 42 cases in the study, 8 cases (19%) had a Ki67 proliferation index that didn’t correlate with their stage and grade at presentation . In the low grade and stage, 5 cases had a high proliferative index, 2 cases in the higher stage had a low proliferative index and 1 case of mucinous adenocarcinoma had a very
87 high proliferative index.The prognosis and biological behavior of these cases could vary from the other cases in the same stage and grade.
Immunohistochemical study of Ki67 proliferation index in cases of colorectal carcinoma was found to provide an additional clue regarding the biological behavior of a significant number of tumours and could help in predicting the prognosis of colorectal carcinomas.
88 FIGURE (i): PHOTOMICROGRAPH SHOWING KI67 IMMUNOSTAINING IN A NORMAL INTESTINAL MUCOSA (100X) FIGURE (ii): PHOTOMICROGRAPH SHOWING WELL-DIFFERENTIATED ADENOCARCINOMA,H&E STAIN (100X)
FIGURE (iii): PHOTOMICROGRAPH SHOWING KI67 IMMUNOSTAINING IN A WELL-DIFFERENTIATED ADENOCARCINOMA (100X) FIGURE (iv): PHOTOMICROGRAPH SHOWING MODERATELY- DIFFERENTIATED ADENOCARCINOMA,H&E STAIN (100X)
FIGURE (v): PHOTOMICROGRAPH SHOWING KI67 IMMUNOSTAINING IN A MODERATELY-DIFFERENTIATED ADENOCARCINOMA (100X) FIGURE (vi): PHOTOMICROGRAPH SHOWING POORLY-DIFFERENTIATED ADENOCARCINOMA,H&E STAIN (100X)
FIGURE (vii): PHOTOMICROGRAPH SHOWING KI67 IMMUNOSTAINING IN A POORLY-DIFFERENTIATED ADENOCARCINOMA (100X) FIGURE (viii): PHOTOMICROGRAPH SHOWING MUCINOUS ADENOCARCINOMA,H&E STAIN (100X)
FIGURE (ix): PHOTOMICROGRAPH SHOWING KI67 IMMUNOSTAINING IN A MUCINOUS ADENOCARCINOMA (100X) BIBLIOGRAPHY
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S.N Ki67 O. PATH NO. AGE SEX IP NO SITE CLASSIFICATION GRADE SEROSA NODES TNM STAGE INDEX %
1 9/18 41 F 94740 ASCENDING COLON ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 1 / 5 pT4aN1a III B 25.5 2 34/18 63 F 94864 SIGMOID COLON ADENOCARCINOMA MOD.DIFFERENTIATED FREE 10/11 pT3N2b III C 67.9 3 60/18 40 M 90356 ASCENDING COLON ADENOCARCINOMA POOR.DIFFERENTIATED INVOLVED 0/12 pT4bN0 II C 59.1 MUCINOUS 4 209/18 48 M 1844 CAECUM ADENOCARCINOMA MUCINOUS INVOLVED 0/8 pT4bN0 II C 37.8 5 393/18 56 F 95482 TRANSVERSE COLON ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/21 pT3N0 II A 63 6 586/18 53 M 6038 RECTUM ADENOCARCINOMA MOD.DIFFERENTIATED FREE 0/5 pT2N0 I 59.4 7 642/18 45 M 5334 ASCENDING COLON ADENOCARCINOMA POOR.DIFFERENTIATED FREE 1 / 2 pT3N1a III B 59.5 8 789/18 72 F 16301 SIGMOID COLON ADENOCARCINOMA MOD.DIFFERENTIATED FREE 0/5 pT3N0 II A 65.8 9 819/18 61 F 14477 RECTUM ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 0/1 pT4aN0 II B 52.1 10 1113/18 75 F 15688 TRANSVERSE COLON ADENOCARCINOMA MOD.DIFFERENTIATED FREE 0/10 pT3N0 II A 38.1 11 1312/18 46 M 24249 RECTUM ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 3 /13 pT4aN1b III B 71.4 MUCINOUS 12 434/17 51 F 4389 ASCENDING COLON ADENOCARCINOMA MUCINOUS FREE 0/3 pT3N0 II A 30.3 SQUAMOUS CELL 13 561/17 52 F 8816 SIGMOID COLON CARCINOMA INVOLVED 0/1 pT4bN0 II C 60.1 14 686/17 58 M 11605 RECTO-SIGMOID JUNCTION ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/3 pT4bN0 II C 54.6 15 1415/17 60 M 26536 CAECUM ADENOCARCINOMA wELL DIFFERENTIATED FREE 0/3 pT3N0 II A 38.9 MUCINOUS 16 1999/17 17 F 39794 DESCENDING COLON ADENOCARCINOMA MUCINOUS INVOLVED 4 / 8 pT4aN2a III C 24.6 17 2105/17 76 M 36512 HEPATIC FLEXURE ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/10 pT3N0 II A 47.8 18 2176/17 55 F 41989 SIGMOID COLON ADENOCARCINOMA MOD.DIFFERENTIATED FREE 0/1 pT3N0 II A 46.8 19 2537/17 65 F 46069 RECTUM ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/12 pT3N0 II A 64.2 20 2821/17 55 M 46264 ASCENDING COLON ADENOCARCINOMA WELL DIFFERENTIATED INVOLVED 0/2 pT4aN0 II B 65.6 21 3032/17 52 F 57470 TRANSVERSE COLON ADENOCARCINOMA WELL DIFFERENTIATED INVOLVED 1 /11 pT4aN1a III B 63.2 22 3076/17 51 F 57426 DESCENDING COLON ADENOCARCINOMA WELL DIFFERENTIATED INVOLVED 0/1 pT4aN0 II B 66.8 23 3636/17 58 M 68391 CAECUM ADENOCARCINOMA WELL DIFFERENTIATED INVOLVED 0/5 pT4aN0 II B 36.5 24 3935/17 65 F 67517 RECTUM ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/4 pT2N0 I 63.9 25 3995/17 36 M 71206 RECTUM ADENOCARCINOMA WELL DIFFERENTIATED INVOLVED 3/4 pT4aN1b III B 64.7 26 966/16 70 M 18725 SIGMOID COLON ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 0/4 pT4aN0 II B 24.3 27 1916/16 60 F 31548 RECTUM ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/3 pT3N0 II A 38 28 2304/16 50 M 42898 DESCENDING COLON ADENOCARCINOMA WELL DIFFERENTIATED FREE 0/6 pT3N0 II A 40.2 MUCINOUS 29 3159/16 50 F 56255 CAECUM ADENOCARCINOMA MUCINOUS FREE 2 /9 pT3N1b III B 23.7 MUCINOUS 30 3244/16 73 M 51221 RECTO-SIGMOID JUNCTION ADENOCARCINOMA MUCINOUS INVOLVED 0/1 pT4bN0 II C 35 MUCINOUS 31 3662/16 35 M 63557 SIGMOID COLON ADENOCARCINOMA MUCINOUS FREE 5/16 pT3N2a III B 67.5 32 3730/16 72 F 64873 CAECUM ADENOCARCINOMA MOD.DIFFERENTIATED FREE 2 /8 pT2N1b III A 34.8 33 3785/16 46 M 69135 ASCENDING COLON ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 1 /15 pT4bN1a III C 74.6 34 4071/16 65 M 70893 RECTUM ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 0/3 pT4aN0 II B 41.3 35 4131/16 60 F 73233 ASCENDING COLON ADENOCARCINOMA POOR.DIFFERENTIATED INVOLVED 15/15 pT4aN2b III C 73.4 36 4306/16 40 M 75200 RECTUM ADENOCARCINOMA POOR.DIFFERENTIATED INVOLVED 1 /6 pT4aN1b III B 63.8 37 1179/15 50 M 20193 RECTO-SIGMOID JUNCTION ADENOCARCINOMA MOD.DIFFERENTIATED FREE 1 /9 pT3N1a III B 50.8 38 1861/15 60 M 31655 SIGMOID COLON ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 0/3 pT2N0 I 62.1 MUCINOUS 39 2626/15 75 F 45129 ASCENDING COLON ADENOCARCINOMA MUCINOUS INVOLVED 6 /6 pT4aN2a III C 14.5 40 2963/15 24 F 50770 CAECUM ADENOCARCINOMA WELL DIFFERENTIATED INVOLVED 0/2 pT4bN0M1c IV C 50.8 41 3297/15 40 F 56246 RECTUM ADENOCARCINOMA MOD.DIFFERENTIATED INVOLVED 1 /2 pT4aN1a III B 53.5 42 3303/15 45 F 66208 SIGMOID COLON ADENOCARCINOMA MOD.DIFFERENTIATED FREE 0/2 pT3N0 II A 45 LIST OF ABBREVIATION
AJCC: American Joint Committee on Cancer
APC: Adenomatous polyposis coli
BAX: Bcl-2–associated X protein
Bcl-2: B-cell lymphoma-2
BrdU: 5-Bromodeoxyuridine
BrdUrd: Bromodeoxyuridine
CDKs: Cyclin-dependent kinases
CRC: Colorectal carcinoma
DAB: Diaminobenzidine
DCC: Deleted in colon cancer
DNA: Deoxy ribonucleic acid
FAP: Familial adenomatous polyposis coli
FOBT: Fecal occult blood test
G1 phase: Gap 1 phase
HNPCC: Hereditary non polyposis colorectal cancer
HPF: High-power fields
IGF2: Insulin-like growth factor 2
JP: Juvenile polyposis
LI: Labeling index LOH: Loss of heterozygosity,
MAP kinase: Mitogen-activated protein kinase
MMP7: Matrix metalloproteinase 7
MMR: Mismatch Repair
MSI: Microsatellite instability
MSI-H: High-frequency MSI
MSI-L: Low-frequency MSI
MSS: Microsatellite stable
P value: Probability factor
PBS: Phosphate buffered saline
PCNA: Proliferating cell nuclear antigen
SEER: Surveillance, Epidemiology and End Results
SPSS: Statistical Product for Services Solutions
TGF-β: Transforming growth factor beta
TGFβR-II: Transforming growth factor beta receptor-II
WHO: World Health Organization