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CLINICOPATHOLOGIC PATTERN AND DNA METHYLATION
STATUS OF COLORECTAL CARCINOMA IN A GROUP OF
BANGLADESHI PATIENTS
Dr. Abu Khalid Muhammad Maruf Raza
MBBS
Contents Page
_________________________________________________ no
1 INTRODUCTION 01
2 REVIEW OF LITERATURE
a Anatomical consideration 04
b Physiology 08
c Tumors of the colon and rectum 09
3 MATERIALS AND METHODS 26
4 OBSERVATIONS AND RESULTS 33
5 DISCUSSION 54
6 SUMMARY AND CONCLUSION 62
7 BIBLIOGRAPHY 64
8 APPENDICES
I Certificate from The Chicago University i
II Clinical proforma ii
III Preparation of stains and chemicals iv
IV Tissue processing and staining procedures v
V Protocol for DNA extraction viii
VI Protocol for DNA methylation x
VII TNM staging system xii
VIII Master table xiv
IX Illustrations xviii
LIST OF TABLE
Table
no
Title Page no
2.3:1 Genes silenced by hypermethylation in colorectal cancer 18
2.3:2 TNM Classification of Colorectal Carcinoma by American Joint
Committee on Cancer (AJCC).
22
2.3:3 TNM staging for colorectal carcinoma. 23
4.1 Sex distribution in relation to age group. 35
4.2 Symptoms with tumour location of colorectal cancer cases. 36
4.3 Haemoglobin concentration in 50 cases of colorectal cancer
patients
37
4.4 CEA (Carcinoembryonic antigen) level in 50 colorectal
cancer cases
37
4.5 Site distribution of colorectal cancer from distal to proximal 38
4.6 Histological diagnosis of 50 cases of colorectal cancer 40
4.7 Histological grading of colorectal cancer cases with sex and
location
41
4.8 TNM (Tumour, Node, Metastasis) stage with sex and location 42
4.9 Signet ring cells in colorectal cancer patients 45
4.10 Circumferential margin involvement, Perineural invasion and
Tumour border configuration with TNM stage.
47
4.11 Selected genes that show methylation differences in 24 cases of
Colorectal carcinoma.
53
5.1 Age and sex distribution of CRC in different studies and the
present study.
54
5.2 Distribution of colorectal cancer in left colon and right colon. 57
LIST OF FIGURES
Fig.
No
Title Page
no
2.1 Gross anatomy of large intestine 07
4.1 Bar diagram showing age distribution of colorectal cancer. 34
4.2 Growth pattern of colorectal cancer cases. 39
4.3 Various grade of tumour infiltrating lymphocytes in 50 cases. 43
4.4 Lymphovascular invasion in colorectal cancer patients 44
4.5 Extracellular mucin in colorectal cancer patient. 45
4.6 Scatter diagram showing total intensity in tumour tissue and its
corresponding normal tissue in all 24 cases
48
4.7 Scatter diagram showing average beta of all 48 samples 49
4.8 Histogram showing tumour differential score. 50
4.9 Bar diagram showing sources of variation of methylation data. 51
4.10 Variation of methylation data due to tumour differentiation. 52
1.0 INTRODUCTION
Colorectal cancer (CRC) is the third most common cancer in the world and the second
leading cause of cancer related deaths in the United States. Globally, the incidence of
CRC varies widely with higher incidence rates in North America, Australia and Northern
and Western Europe (Aljebreen, 2007). The lifetime risk of developing CRC is about 6%
or one in 18. Over 95% of these CRC is adenocarcinoma (Kim et al, 2010).
CRC is relatively uncommon in Indian sub continent. In India the incidence of colorectal
cancer was found to be 4.2 and 3.2 per hundred thousand for male and female population
respectively (Afroza et al, 2007).
The incidence of colorectal cancer in Bangladesh is not exactly known, it appears to be
common and occur in younger age group with slight male preponderence. Average age at
diagnosis is 10 years less than the developed countries. Rectal bleeding is the most
common symptoms and majority of the carcinoma were in the rectum (Hossain, 2007).
The peak incidence of colorectal carcinoma is in between the age of 60 and 69 years.
Fewer than 20% occur before the age of 50 years. Males are affected slightly more than
females (Turner, 2010). Colorectal carcinoma are rare before the age of 40 without
genetic predisposition or without predisposing factors (Hamilton, 2000).
Early detection of colonic cancers is a challenging task as because clinical symptoms
develop slowly. Per rectal bleeding is common. Many patients experience change in bowel
habit (Yawe et al, 2007). Screening tests like digital rectal examination, simple laboratory
1
investigations like estimation of CEA, estimation of haemoglobin, faecal occult blood test,
and visualization of the gut mucosa by sigmoidoscopy and colonoscopy examination may
be a help in the diagnosis (Aljebreen, 2007).
Colorectal cancer is a multifactorial disease process. Etiology contributing from
environmental factors including diatery factors, obesity, alcohol intake, smoking, life style
and genetic and epigenetic abnormalities. The molecular events that leads to CRC is
heterogenous and includes genetic and epigenetic abnormalities. Genetic events in
colorectal cancer is genetic alteration of the APC gene, mutations in the KRAS and P53
gene and abnormalities in the DNA mismatched repair genes (Turner, 2010).
Epigenetic changes, which is the heritable changes in gene function that are not due to
changes in the DNA sequence is an important pathway in the mechanism of tumerogenesis
in colorectal cancer. DNA methylation abnormalities is an important epigenetic changes in
CRC and become an area of great interest in the field of cancer research. The significance
of DNA methylation alteration in CRC has been reported widely. There is both
hypermethylation and hypomethylation abnomalities in various genetic loci in CRC (Kim
et al, 2010).
Available data indicates some differences in CRC in Bangladeshi population like lower
mean age of patients, predominently left sided tumour (Hossain, 2007). Correlation of
histopathological features with clinical data and detection of genetic abnormalities can give
better insight in this field.
2
1.1 AIM
To see the morphologic pattern, clinical features and DNA methylation changes in
colorectal carcinoma in a group of Bangladeshi patients.
1.2 OBJECTIVES
1. To see various histologic features of colorectal carcinoma.
2. To see different clinical presentation of colorectal carcinoma.
3. To see DNA methylation changes in colorectal cancer tissue and corresponding
surrounding normal colonic tissue.
3
2.0 REVIEW OF LITERATURE
2.1 Anatomy of large intestine
2.1.1 Gross anatomy:
The large intestine is about 1.5 meters long, extends from the distal end of ileum to anus.
It begins in the right iliac fossa as the caecum, from which the vermiform appendix arises.
The caecum becomes the ascending colon which passes upwards in the right lumbar region
and hypochondrium to the inferior aspect of the liver. It then bends to the left forming the
hepatic flexure (right colic flexure) and becomes the transverse colon. This loops across
the abdomen with an anterior-inferior convexity until reaches the left hypochondrium.
Where it curves inferiorly to form splenic flexure (left colic flexure) and becomes the
descending colon. It then forms sigmoid colon in the left illac fossa. The sigmoid colon,
descends deep into the pelvis and becomes the rectum, which ends in the anal canal at the
level of pelvic floor (Borley, 2008).
2.1.2 Blood supply of the colon:
Arterial supply: The arteries which supply the parts of the large intestine are from the
colic branch of the superior mesenteric artery. The hind gut derivatives are supplied by the
inferior mesenteric and rectal arteries (Borley, 2008).
4
The venous drainage: The vein of the large gut are the corresponding superior and
inferior mesenteric artery. The rectum and anal canal are drained by the rectal vein
(Borley, 2008).
Lymphatic drainage: The large gut is drained to the epicolic nodes, paracolic nodes, the
intermediate colic and terminal nodes (Borley, 2008).
2.1.3 The nerve supply:
The sympathetic supply of mid gut derivate are coeliac and mesenteric ganglia and the
parasympathetic supply is by the vagus nerve. The hind gut derivatives are supplied by the
superior hypogastric plexus. The rest of the portion is supplied by inferior hypogastrci
plexus and pudendal nerve (Borley, 2008).
2.1.4 Developmental anatomy of colon:
The caecum, the appendix, the ascending colon and the proximal two-third of the
transverse colon are derived from the midgut. The distal one-third of the transverse colon,
the descending colon, the sigmoid colon, the rectum and the upper portion of the anal canal
are derived from hindgut (Sadler, 1995).
2.1.5 Histology of the colon:
The large intestine is divided topographically into three segments ( Junquira et al, 1995):
5
a. Colon,
b. Rectum and
c. Anal canal.
All the segments histologically have four coats;
a. Mucosa
b. Submucosa
c. Muscularis externa and
d. Serosa (Young and Heath, 2000).
The surface epithelium of the mucosa is made of tall columnar epithelium mixed with
goblet cells turn downward as a tubular gland. The lower portions of the tubular glands
have numerous goblet cells. The lamina propria is composed of fibrocollagenous tissue.
The submucosa is composed of loose connective tissue containing large blood vessels and
meissner plexus of nerve.
Muscularis externa in the colon shows some variation from the other parts of the gut. The
inner circular layer is complete and prominent. The outer longitudinal layer forms three
equidistant, longitudinal bands (Tinia coli).
Adventitia or serosa consists of thin connective tissue layer covered by the mesothelium
(Yound and Heath, 2000).
6
7
2.2 Physiology of the large intestine
The main function of the colon is absorption of water, Na+, and other minerals by removal
of about 90% of the fluid. It converts the 1000-2000ml of isotonic chyme that enters it
each day from the ileum to about 200-250ml of semisolid feces (Ganong, 2005).
Motility and secretion of the colon: When peristaltic wave reaches to the valve, it opens
and permits ilial chyme to squirt into the caecum. The movement of the colon include
segmentation contraction and peristalsis. Segmentation contraction mixes the content of
the colon and by exposing the more of the contents to the mucosa facilitates absorption.
Peristaltic waves propel the contents towards the rectum (Ganong, 2005).
Transit time in the colon: The first part of the test meal reaches the caecum in about 4
hours and all of the undigested food entered the colon in 8 to 9 hours. The first meal
reaches the pelvic colon in 12 hours (Ganong, 2005).
Absorption in the colon: Na+is actively transported out of the colon and water follows
along the osmotic gradient. K+and HCO3 are secreted into the colon. The absorptive
capacity of the colon makes it a suitable route of drug administration (Ganong, 2005).
Daefecation: Distention of the rectum with faces initiates reflex contraction of the
musculature and desire to defecate. The sympathetic nerve supply to internal anal sphincter
is excitatory whereas the parasympathetic nerve supply is inhibitory (Ganong, 2005).
8
2.3 Tumours of the colon and rectum
Many conditions, such as congenital disease, infection, inflammatory diseases, motility
disorders and tumours affect the large intestine (Turner, 2010). The colorectal cancer is
the second most common visceral cancer in the U.S.A. (Cooper, 1999). Virtually 98% of
colorectal carcinoma are adenocarcinoma (Turner, 2010). The World Health Organization
classified colorectal cancer histologically as follows (Hamilton. 2000).
2.3.1 WHO classification of tumors of colon and rectum: (Hamilton, 2000)
1. Non-neoplastic polyp
i. Hyperplastic polyp
ii. Peutz-Jeghers polyp
iii. Juvenile polyp
2. Epithelial tumors
1. Adenoma
i. Tubular adenoma
ii. Villous adenoma
iii. Tubulovillous adenoma
iv. Serrated adenoma
9
2. Intraepithelial neoplasia (Dysplasia) associated with Chronic inflammatory
diseases
Low-grade glandular intraepithelial neoplasia
High grade glandular intraepithelial neoplasia
3. Carcinoma
i. Adenocarcinoma
ii. Mucinous carcinoma
iii. Signet ring carcinoma
iv. Small cell carcinoma
v. Adenosquamous carcinoma
vi. Medullary carcinoma
vii. Squamous carcinoma
viii. Undifferentiated carcinoma
4. Carcinoid tumour
5. Mixed carcinoid-adenocarcinoma
6. Non-epithelial tumor
i. Lipoma
ii. Leiomyoma
iii. Gastrointestinal stromal tumor
10
iv. Leiomyosarcoma
v. Angiosarcoma
vi. Malignant lymphoma
vii. Malignant melanoma
7. Secondary tumors
2.3.2 Colorectal carcinoma
Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide and the
third most common malignancy and one of the leading causes of cancer death in women
and men in the United States. The lifetime risk of CRC among women and men at average
reaches 6% or one in 18 (Kim, 2010). The peak incidence of colorectal carcinoma is
between 60 and 79 years. Fewer than 20% cases occur before the age of 50 years (Turner,
2010).
2.3.2.1 Incidence of colorectal carcinoma
Colorectal cancer (CRC) is the third most common cancer and the second leading cause of
cancer related death in the United States (Aljebreen, 2007). In the USA and Western
Europe, colorectal cancer constitutes approximately 10% of all malignancies (Leon et al,
2004). It represents 9.4% of all incident of malignancy in men and 10.1% in women
globally. CRC is not equally common through out the world. The incidence varies in
various geographical part of the world. High incidence seen in Europe, America,
Australia, New Zealand with incidence ranges from 25.3/100000 to 45.8/100000
11
population (Boyle and Langman, 2000). Compare to developed countries, lower incidence
of colorectal cancer is seen in developing countries like Africa and Asia, including central
and south Asia and India. Incidence ranges from 2/100000 to 8/100000 population
(Notani, 2001). The epidemiological study of colorectal cancer done by Keating et al
(2003) among 2272 pathological report in New Zealand showed the incidence of colorectal
carcinoma was 46.8/1000000 for men and 43.5/100000 for women. Ayyub et al (2000), in
a large hospital of Saudi Arabia has shown the incidence were 4.6/100000 in male and
4.4/100000 in female population.
2.3.2.2 Age and Sex distribution of Colorectal carcinoma
Colorectal cancer is a disease of advanced aged population. Greater number of CRC are
found in the six decades with a median age of about 62 years (Riddell, 2003). Keating et
al (2003) done a study on the epidemiology of colon cancer in New Zealand. They found
in their study that the mean age of women was 69.4 years and mean age of men was 68.5
years. Turner (2010) have shown nearly the same result in their study as the peak
incidence of colorectal carcinoma was in between ages 60-70 and only 20% cases occur
before the age of 50 years. Ayyub et al (2002) studied on clinicopathological trends in
colorectal cancer on 160 cases in Saudi Arabia. They showed that the mean age was
56.3±14.98 years.
Colorectal cancer is extremely rare in paediatric age group. Afroza et al (2007) reported a
11 year old Bangladeshi boy with primary mucinous adenocarcinoma in the rectum.
12
Colorectal cancer affects men and women almost equally (Boyle and Langman 2000).
Some study have shown male predominance (riddell et al, 2003). Keating et al (2003)
showed in their study that the male and female ratio were almost equal. Fireman et al
(2005) showed female predominance in their study as among 624 cases, 271 were male
and 353 were female.
2.3.2.3 Clinical features of Colorectal carcinoma
Patients with colorectal cancer have usually presented with abdominal pain, alteration of
bowel habit, loss of weight, vomiting, frequently with colic, anorexia, bleeding per rectum,
lump, indigestion and acute on chronic obstruction (Hamilton, 2000). Ayyub et al (2002)
mentioned on their study that most of the patient presented with the symptom of abdominal
pain, altered bowel habit, per rectal bleeding weight loss, intestinal obstruction and
constipation. Duration of symptom varied from one month to 2 years.
2.3.2.4 Investigations in CRC
The laboratory investigation include the blood and biochemical parameters like tumour
markers (CEA, CA-19-9), and also the visualization of the lesion through endoscope.
Ayyub et al (2002) studied clinicopathological trends in colorectal carcinoma showed
anaemia (Hb% lOgm/dl or less) in 55% of the patients. The distribution of anaemia varied
significantly in various sub site of primary colorectal carcinoma. About 30% patients had
anaemia with left sided tumour and 70% of patients in right side colon cancer. They also
measure CEA level in serum of colorectal cancer patients. CEA level varied from 1 to
13
850.2 with a mean of 48.62. Leon et al (2004) studied trend of incidence, subtype
distribution and staging of colorectal carcinoma in the 15 years experience of a specialized
cancer registry, showed more then 50% colorectal carcinoma can be detected by
sigmoidoscopy and rest needed pancolonscopy for the choice of screening for individual at
the risk of colorectal carcinoma.
2.3.2.5 Sub site distribution
The right colon is considered from caecum to spleenic flexure; left colon includes
descending colon, sigmoid colon and rectum (Gomez et al, 2004). The sub site-specific
variation is seen in the development of colorectal carcinoma. Two-third of colorectal
carcinoma occur in the left colon and the rest in the right colon (Leon et al, 2004). Ayyub
et al (2002) showed majority of the colorectal carcinoma 68.2% in the rectum and sigmoid
colon; and 22.5% colorectal carcinoma originating from ascending and transverse colon.
Gomez et al (2004) showed total 31% of the cancer were in right side. The study also
cited the differences in the anatomical distribution between sex, in female 48% were in
right and 41% were left sided colon cancer in comparison to 59% cases with left colon
cancer and 52% cases with right colon cancer in male.
2.3.2.6 Histological subtypes of colorectal carcinoma
Most of the colon cancer is adenocarcinoma. In the study of Keating et al, (2003)
showed 94.7% cases of adenocarcinoma, 4.2% were mucinous adenocarcinoma, 0.4%
were signet ring cell carcinoma and less then 1% cases were adenosquamous, squamous
and miscellaneous carcinoma. Hossain (2007) found 74% of cases were usual
14
adenocarcinoma, 23% cases mucinous adenocarcinoma, 1.5% cases each signet ring cell
carcinoma and small cell carcinoma.
2.3.3 Pathogenesis
Environmental and genetic factors both contribute to colorectal cancer development.
Genetic susceptibility may be the soil on which subsequent environmental factors act
(Riddell, 2003).
2.3.3.1 Environmental factors:
Epidemiologic studies have indicated that meat consumption, smoking, sedentary lifestyle
and alcohol consumption are risk factors for colorectal carcinoma. Inverse associations
include vegetable consumption, prolonged use of non-steroidal anti inflammatory drugs,
oestrogen replacement therapy and physical activity (Hamilton, 2000).
2.3.3.2 Molecular carcinogenesis:
The combination of molecular events that lead to colonic adenocarcinoma is heterogenous
and includes genetic and epigenetic abnormalities (Turner, 2010). The genetic and
epigenetic pathways involved are:
1. -catenin pathway which is associated with adenoma-carcinoma sequence.
2. Loss of p53.
3. K-ras mutation.
4. Delated in colon cancer gene (DCC gene).
15
5. Telomerase activity.
6. Microsatellite instability pathway which is associated with DNA mismatch repair.
7. DNA methylation abnormalities (Hypomethylation and Hypermethylation).
2.3.3.3 DNA methylation in cancer
Cancer results from the accumulation of mutation in the genes. In addition to genetic
mutation, this epigenetic change has been included as an alternative mechanism to cancer
development. Epigenetics refers to change in the pattern of gene expression by
mechanisms other than alterations in the primary nucleotide sequence of a gene(Herman,
2003). DNA methylation is the enzymatic attachment of methyl group to the 5th
carbon of
the cytosine base (Samarakoon, 2010). Methylation usually occurs in the CpG islands, a
cytosine guanosine rich region in the DNA. In humans, DNA methylation is carried out by
a group of enzymes called DNA methyltransferases. The letter “p” here signifies that the C
and G are connected by a phosphodiester bond. Methylation patterns in tumor cells are
significantly different from those in normal cells (Herman, 2003).
Silencing or inactivation of tumour suppressor gene is seen in cancers mostly due to
hypermethylation. Hypermethylation profiling over more than 15 tumor types (colon,
stomach, pancreas, liver, kidney, lung, breast, ovary, endometrium, kidney, bladder, brain,
and leukemia and lymphomas) has shown that all the metabolic pathways are affected by
promoter hypermethylation-associated silencing. Hypomethylation leads to the
inappropriate and increased levels of gene expression in tumors such as oncogene
activation (Samarakoon, 2010).
16
2.3.3.4 DNA methylation in colorectal cancer:
Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and
epigenetic alterations in colonic epithelial cells during neoplastic transformation (Kim,
2010).
Hypermethylation in the CpG islands of the tumor suppressor gene promoters can lead to a
complete block of transcription and inactivates the tumor suppressor genes. DNA
hypomethylation could also drive neoplastic progression and transformation. It may make
chromosomes more susceptible to breakage and oncogene activation (Kim, 2010).
2.3.3.4.1 Genes inactivated by promotor hypermethylation in colorectal cancer
Hypermethylated genes are associated with colorectal neoplasia includes the tumour
suppressor, mismatch-repair and cell-cycle regulatory genes (Wong, 2007). Table 2.3.1
shows functions and frequency of different genes involved by hypermethylation in
colorectal cancer.
17
Table: 2.3.1 Genes silenced by hypermethylation in colorectal cancer (Wong, 2007)
Genes Function Frequency (%)
APC Signal transduction, beta-catenin regulation 10–50
CDH13 Cell signalling (cell recognition and adhesion) 30–40
CDKN2A Cell-cycle regulation 15–30
CHFR Mitotic stress checkpoint 30–40
HIC1 Regulation of DNA damage responses 80
HPP1 Transmembrane transforming growth factor
(TGF)- antagonist
80
LKB1 Cell signalling, cell polarity 5–10
MGMT Repair of DNA guanosine methyl adduct 30–40
MLH1
Genes
Mismatch repair
Function
10–20
p14ARF Cell-cycle regulation 20–30
RASSF1A DNA repair, cell-cycle regulation >50
SOCS1 Cell signaling 5–10
THBS1 Angiogenesis 10–20
TIMP3 Matrix remodelling, tissue invasion 10-30
18
2.3.3.4.2 DNA hypomethylation in colorectal cancer
Hypomethylation may make chromosomes more susceptible to breakage and therefore lead
directly to genomic instability. DNA hypomethylation can also lead to the activation of
oncogenes such as S100A4 metastasis-associated gene in colorectal carcinoma and can
lead to loss of imprinting (LOI) which can drive cellular proliferation in cancer. The
clearest example of this phenomenon is hypomethylation of IGF2/H19 seen in about 40%
of colorectal cancer tissue (Wong, 2007). Hypomethylation of CDH3 promotor, CD133,
LINE-1 are also seen in colorectal cancer (Kim, 2010).
2.3.4 Gross morphology of colorectal carcinoma
The tumor of the proximal colon tends to grow as polypoid and exophytic mass and in the
distal colon the mass grow as annular and encircling manner. The gross morphology of the
lesions are fungating. annular, tubular and ulcerated. The fungating growth is cauliflower
like growth, some time ulceration is seen in the tip of the fungating growth. The annular
growth encircling whole of the circumference and produce obstructive feature clinically.
The tubular growth is flat (linitis plastica). The ulcerated growth form an ulcer in the gut
wall. The superficial spreading lesion is difficult to identify grossly (Turner, 2010). The
cut surface of the gut wall shows grayish white tissue replacing the bowel wall. Highly
mucinous tumour have a gelatinous glaring appearance, and layers of mucus may separate
the layers of the bowel wall (Turner, 2010).
19
2.3.5 Microscopic features of colorectal carcinoma
2.3.5.1 Adenocarcinoma: About eighty percent of colorectal carcinomas are histologically
characterized by good gland formation and varying degrees of differentiation, from well to
moderately differentiated. Well differentiated carcinoma have >95% glandular structure,
moderately differentiated carcinoma have 50-95% glands, poorly differentiated carcinoma
have 5-50% glandular structure and undifferentiated carcinoma have <5% glandular
structures (Hamilton 2000)
2.3.5.2 Mucinous adenocarcinoma: Mucinous and Signet-ring adenocarcinoma account
for 10% of colorectal cancer. This designation is used if >50% of the lesion is composed of
mucin. This variant is characterized by pools of extracellular mucin that can contain
malignant epithelium as acinar structure, strips of cells or single cells (Hamilton,2000)
2.3.5.3 Signet ring cell adenocarcinoma: Signet-ring and mucinous cell carcinoma
account for approximately 10% of colorectal cancer. This variant of adenocarcinoma is
definied by the presence of >50% of the tumour cells with prominant intracytoplasmic
mucin (Hamilton, 2000)
2.3.5.4 Small cell carcinoma: A rare variant is small cell carcinoma which composes <1%
of colorectal cancer (Cooper, 1999)
2.3.5.5 Adenosquamous carcinoma: These unusual tumours show features of both
squamous carcinoma and adenocarcinoma either as separate areas within the tumour or
admixed pattern (Hamilton, 2000).
20
2.3.5.6 Squmous cell carcinoma: Squamous cell carcinoma of the colon is very rare. To
make diagnosis of the squamous cell carcinoma in the colon, there must be no other site of
the squamous cell carcinoma in the body and no involvement of cloacogenic squamous
lined mucosa (Rosai, 2004).
2.3.5.7 Medullary carcinoma: This rare variant is characterized by the sheets of
malignant cells with vesicular nuclei, prominent nucleoli and abundant pink cytoplasm
exhibiting prominent infiltration by intraepithelial lymphocytes (Hamilton, 2000).
2.3.5.8 Undifferentiated cancer: Undifferentiated cancer is uncommon, accounting for
approximately 1 % of the colorectal carcinoma. They are malignant epithelial neoplasm
that have no glandular structure or other features to indicate definite differentiation
(Hamilton, 2000).
2.3.6 Staging of colorectal cancer:
The major role of proper staging of colorectal carcinoma is to provide information to
physician regarding patient's prognosis and the need for adjuvant therapy. For many years,
pathologists used the classic Dukes' classification(1932), Astler-Coller classification(1954)
and the TNM classification(Turner, 2010). Table 2.3.2 shows TNM classification and
Table 2.3.3 shows TNM staging of colorectal carcinoma (Turner, 2010).
21
Table:2.3.2 TNM Classification of Colorectal Carcinoma by American Joint
Committee on Cancer (AJCC) (Turner, 2010).
TUMOUR
Tis In situ dysplasia or intramucosal carcinoma
T1 Tumor invades submucosa
T2 Tumor invades into, but not through, muscularis propria
T3 Tumor invades through muscularis propria
T3a Invasion <0.1 cm beyond muscularis propria
T3b Invasion 0.1 to 0.5 cm beyond muscularis propria
T3c Invasion >0.5 to 1.5 cm beyond muscularis propria
T3d Invasion >1.5 cm beyond muscularis propria
T4 Tumor invades adjacent organs or visceral peritoneum
T4a Invasion into other organs or structures
T4b Invasion into visceral peritoneum
REGIONAL LYMPH NODES
NX Lymph nodes cannot be assessed
N0 No regional lymph node metastasis
N1 Metastasis in one to three regional lymph nodes
N2 Metastasis in four or more regional lymph nodes
DISTANT METASTASIS
MX Distant metastasis cannot be assessed
M0 No distant metastasis
M1 Distant metastasis or seeding of abdominal organs
22
Table :2.3.3 TNM staging for colorectal carcinoma (Turner, 2010)
Stage-I Tl N0 M0 or T2, N0, M0
Stage-II T3 N0 M0 or T4, N0, M0
Stage-III T any Nl M0 or T any,N2,N3, M0
Stage-IV T any N any Ml
2.3.7 Grading of colorectal cancer:
Grading is done on the basis of differentiation of tumour cells. Differentiation refers to the
extent to which neoplastic cells resemble comparable normal cells (Kumar et al, 2010).
The formation of glands (acini) is the basis for grading system in CRC. Well differentiated
carcinoma have >95% glandular structure, moderately differentiated carcinoma have 50-
95% glands, poorly differentiated carcinoma have 5-50% glandular structure and
undifferentiated carcinoma have <5% glandular structures. Mucinous carcinoma and signet
ring cell carcinoma by definition are poorly differentiated cancer (Hamilton, 2000).
2.3.8 Prognosis of colorectal cancer: (Rosai, 2004)
There are several clinical and pathological parameters which determines prognosis. Most
important is the tumour stage. 5 years survival rate in TNM stage I disease is 90 to 98
percent. About 80 percent 5 year survival in TNM stage II and less than 10 percent in
stage III with many lymph nodes involvolvement and in stage IV (Riddell, 2003). Some
factors related to prognosis are given below:
23
Age: Tumour occuring in very young and old patient have poor prognosis.
Sex: The prognosis is better in female than in male.
CEA serum level: >5.0 ng/ml have been shown to have adverse prognosis.
Tumour location: Tumour located in the sigmoid colon and rectum have the worse
prognosis.
Local extent: Lymph node metastasis have worse prognosis than local invasive.
Tumour size: There is a little relationship between the size of the tumour and prognosis.
Tumour edge: The non-polypoid edge of the tumour have the worse prognosis than
polypoid cancer.
Obstruction: Obstruction is an indicator of worse prognosis.
Perforation: It is due to invasion to the wall shows worse prognosis.
Tumour margin and inflammatory reaction: Pushing margin with inflammatory
response have better prognosis.
Vascular invasion: When venous invasion is present, the 5-years survival decreases
markedly.
Perineural invasion: Reflects the advanced stage of the disease.
Surgical margins: Presence of tumour in surgical margin is the single most critical factor
for recurrence of carcinoma.
24
Tumour thickness: Central depressed area of the tumour is correlated with the presence of
lymph node and liver metastasis and also with bad prognosis.
Microscopic tumour type: Mucinous carcinoma, Signet ring cell carcinoma and
anaplastic carcinoma have worse prognosis.
Acinar morphology: Microacinar pattern of growth is associated with poor prognosis.
Tumour angiogenesis: Tumour angiogenesis predict recurrence and is associated with
decreased survival in colorectal carcinoma.
Mucin-related antigen: Colorectal carcinoma that express the mucin-associated antigens
sailyl-Tn and sailyl-Lewis antigen have more aggressive clinical course.
HLA-DR expression: Patient with tumour having strong HLA-DR expression show better
prognosis.
HCG expression: Effect as adverse indicator of prognosis.
Bcl-2 protein expression: Associated with better prognosis.
DNA ploidy: The DNA aneuploidy and risk of recurrence of survival.
Allelic loss of chromosome 18q: Have strongly negative prognostic significance.
25
3. MATERIAL AND METHOD
3.1 Place and period of study
a. The clinical and histopathological part of this study was carried out at the
Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU),
Dhaka during the period of November 2009 to July 2010. A total of 50 cases of
colorectal adenocarcinoma from all ages and both sexes were included in the study.
b. The DNA methylation part of this study performed on 48 samples (24 tumour tissue
and 24 corresponding healthy tissue) was carried out in collaboration at the Department
of Health Studies, Biological Sciences Division (BSD) of The University of Chicago
Medical Center, Chicago , IL , USA (Appendix-I).
3.2 Collection of the case
A total of 50 cases of colorectal carcinoma were collected for study. For selection,
inclusion criteria described below was followed. Cases were collected from
Bangabandhu Sheikh Mujib Medical University (BSMMU) and private hospitals and
clinics in Dhaka city.
3.3 Inclusion criteria
a. Histologically confirmed cases of adenocarcinoma of the colon.
b. Cases with complete clinical information.
c. Availability of fresh unfixed colorectal cancer specimen.
d. Availability of normal uninvolved colonic tissue away from the tumour.
26
3.4 Exclusion criteria
a. Clinically suspected colorectal carcinoma subsequently proved to be non-malignant
lesions after histological examination.
b. Non Hodgkin lymphoma and other non epithelial tumors of the colon.
c. Cases without clinical data.
d. Formalin fixation or delay in receiving specimen.
3.5 Collection of clinical information
Detailed clinical information was obtained by taking history and recorded in clinical
proforma (Appendix-II). Filling up of the clinical proforma was performed in all cases
either from patient's attendants statement and/or from patients file.
3.6 Collection of the tissue block for histopathological examination
Fresh unfixed specimen were obtained after surgical resection. Large plastic container
with lid were used for collection. Containers were properly labeled with identification
number, name of the patient, address, type of specimen and date and place of operation.
3.6.1 Fixation of sample for Histopathological examination:
After collecting blocks for methylation study (describeb next), the unfixed specimen
were transferred to 10% formalin for overnight fixation. The next morning the
specimen were examined during gross cut up and tissue blocks were taken according to
the following procedure (Rosai, 2004)
27
a. Recording of gross features of the specimen:
i. Specimen type, length of the specimen and amount of mesentery.
ii. Tumour characteristics: Tumor size including thickness, extent around bowel,
tumour configaration(exophytic, infiltrative, ulcerating), presence of necrosis or
hemorrhage, tumour number, extent through bowel wall, serosal involvement,
invasion of adjacent organs and distance of the tumour from the proximal and
distal resection margins.
iii. Number of lymph nodes found and size of the largest node.
b. Sections of tissue blocks:
i. Three blocks from tumour including the serous layer.
ii. One block from tumour with surrounding normal mucosa.
iii. Proximal line of resection.
iv. Distal line of resection
v. Appendix, if included in the specimen.
vi. All the lymph nodes.
vii. Polyp/ Suspicious areas.
3.6.2 Tissue processing for routine histopathological examination:
All the tissue blocks were submitted for routine processing and paraffin
Embedding (appendix-III). The tissue processing and staining was performed following
the standard protocol (appendix-IV). Microscopic examination of routine paraffin
sections stained with haematoxyline and eosin staining method were carried out during
which relevent points were recorded.
28
3.6.3 Routine microscopic examination
Routinely stained sections examined first under low power and then under high power
magnification. The following points were noted during examination:
i. Histologic type of growth.
- Adenocarcinoma(NOS).
- Mucinous.
- Signet ring cell.
- Small cell.
ii. Histologic grade (applicable in adenocarcinoma (NOS) type).
- Well differentiated.
- Moderately differentiated.
- Poorly differentiated.
iii. Extent of invasion.
-Submucosa.
-Muscle coat.
-Serosa.
-Pericolic fat.
iv. Proximal and distal resection margins.
v. Circumferential ( radial) margin.
vi. Lymphatic (small vessel) invasion.
vii. Venous (large vessel)invasion.
viii. Perineural invasion.
ix. Tumor border configaration
- Pushing.
29
- Infiltrative.
x. Lymphocytic response
Grade 0 - <10 lymphocytes per high-power field (HPF).
Grade 1- Tumour infiltrating lymphocytes (TIL) level of 10–50 HPF.
Grade 2 -Tumour infiltrating lymphocytes (TIL) level 50-100 per HPF.
Grade 3- Tumour infiltrating lymphocytes (TIL) level >100 per HPF.
xi. Extracellular mucin content
-No extracellular mucin.
-1-49% extracellular (Tumour cell) mucin.
->50% extracellular (Tumour cell) mucin.
xii. Signet ring cells (Ogino et al, 2008)
- No signet ring cell.
- 1-49% of tumour cell.
- >50% of tumour cells.
xiii. Lymph node status- number of lymph nodes involved.
The tumour was classified according to the World Health Organization classification
(Hamilton, 2000). The tumour staging was done using TNM classification (Turner,
2010).
30
3.7 DNA methylation study
3.7.1 Tissue sample collection for DNA methylation study
Paired unfixed tissue samples, one each from tumour and healthy mucosa were taken in
DNAse free 1.5 ml eppendorf tube (Ambion Catalogue # AM12450, Ambion
company, USA). Dimention of the tissue blocks were 4 to 5 mm. These eppendorf
were kept in -20°c and sent to the department of Health Studies, Biological Sciences
Division of The University of Chicago Medical Center, Chicago , IL , USA in dry ice.
DNA extraction and methylation work done at the Department of health studies (BSD),
The University of Chicago, Chicago, IL, USA under aggrement between the
department of pathology, BSMMU and Department of health studies (BSD), The
University of Chicago, Chicago, IL, USA.
3.7.2 DNA extraction from colonic tissue:
DNA extraction were done using Gentra Purgene Tissue kit (Qiagen Catalogue #
158622, Qiagen company, USA) (Appendix-V).
3.7.3 DNA Methylation by Infinium Assay:
Principle:
The DNA sample is first treated with sodium bisulphite which converts the
unmethylated cytosine bases to uracil. The methylated cytosines on the other hand,
remains unaffected. DNA methylation in this study was based on Infinium assay
31
system using The Human Methylation 27 BeadChip (Inf HumanMeth27, RevB BC
Kit, Catalogue # WG-311-1201 introduced by Illumina Inc, USA) ( Appendix-VI).
3.8 Statistical analysis:
Histopathological portion: All the necessary and relevant data were recorded
methodically and meticulously as far as possible in the clinical proforma. Relevant data
were analyzed by standard statistical method.
DNA methylation portion: The raw image data was processed by Bead Studio
software provided by Illumina inc. USA, to generate the average beta values. Average
beta is the methylation status of a gene locus. It is calculated by intensity of the
methylated sequence(x)/ [intensity of the methylated sequence (x)+ intensity of the
unethylated sequence (y) ]. The ratio is between 0 to 1, where 0= extremely
hypomethylated and 1= extremely hypermethylated. Unpaired t-test was done to
compare the average beta between the tumour & normal tissue. DiffScore was
calculated from the p-value of the un-paired t-test. Delta Beta was calculated as beta of
tumor tissue minus beta of normal tissue.
32
4.0 OBSERVATIONS AND RESULTS
This cross sectional study was undertaken to see the morphologic pattern and DNA
methylation changes in colorectal carcinoma in a group of Bangladeshi patients. Clinical
data were recorded. DNA methylation of non tumorous areas of colon of the same patient
was also determined.
A total of 50 cases of colorectal cancer with their corresponding normal mucosa were
included in the study. As molecular genetic laboratory facility is not available at present in
BSMMU, the DNA methylation part was done in the molecular biology laboratory of the
Department of Health Studies (BSD) of the University of Chicago,Chicago, IL, USA with
aggrement between two department (Appendix-I).
4.1 Age and sex distribution
4.1.1: Age distribution of the colorectal carcinoma cases
The age range was from 19 years to 84 years with a mean age of 46.6 ± 14.8 years. The
patients were divided into 8 groups on the basis of decades. Out of 50 cases maximum
number 12 (24%) of patients belonged to the age group 50-59 years, followed by 11(22%)
cases each in 30-39 years and 40-49 years groups, 6(12%) cases in 20-29 years group, 5
(10%) cases in 60-69 years group, 3 (6%) in 70-79 years group and 1 (2%) case each in
10-19 years and 80-89 year groups. Figure 4.1 shows age distribution of colonic cancer
patient. From this figure, peak age of CRC appears to be 30 to 60 years in our population.
Figure 4.1 : Bar diagram showing age distribution of colorectal cancer.
4.1.2 Male to female ratio
In this study out of total 50 cases, 29 (58%) cases were male and 21 (42%) cases were
female with male to female ratio of 1.4:1, male patients were higher than female patients.
Table 4.1 shows male and female patients with CRC in different age groups.
Table 4.1 : Sex distribution in relation to age group.
Age in years Total number of
cases
No. of Male No. of Female
10-19 1(2%) 1(2%) -
20-29 6(12%) 4(8%) 2(4%)
30-39 11(22%) 5(10%) 6(12%)
40-49 11(22%) 6(12%) 5(10%)
50-59 12(24%) 7(14%) 5(10%)
60-69 5(12%) 3(6%) 2(4%)
70-79 3(6%) 3(6%) -
80-89 1(2%) - 1(2%)
Total=8 50(100%) 29(58%) 21(42%)
4.2 Clinical presentation at the time of attending to doctor
At the time of first consultation, majority of the patients 20(40%) had per-rectal bleeding,
8(16%) had abdominal pain, 7(14%) had altered bowel habit, 6(12%) had generalised
weakness, anorexia and pallor, 6(12%) had combined per rectal bleeding and abdominal
pain and 3(8%) had palpable abdominal mass. The duration of symptoms ranged from one
month to 24 months. Table 4.2 shows clinical presentation of colorectal cancer cases.
Table 4.2: Symptoms with tumour location of colorectal cancer cases.
Symptoms Cases
N=50
Cases in different
location
Right
colon
Left colon
Per rectal bleeding 20 (40%) 1 19
Abdominal pain 8 (16%) 6 2
Altered bowel habit 7 (14%) 4 3
Generalized weakness, anorexia and pallor 6(12%) 4 2
Per rectal bleeding and abdominal pain 6(12%) 4 2
Palpable abdominal mass 3(6%) 2 1
4.3 Haemoglobin concentration in blood
Haemoglobin levels were recorded in 50 cases. It ranged from 8.3 to 14.6 gm/dl.
Haemoglobin level <10gm/dl considered anaemia in both sex (Ayyub,2002).
Haemoglobin was below 10 gm/dl in 13 (26%) cases and in 37(74%) cases it was more
than 10 gm/dl. In 13 cases of below 10gm/dl, 8(62%) were female and 5(38%) were male.
In 37 cases of hemoglobin level more than 10gm/dl, 24(65%) cases were male patient and
13(35%) were female. Table 4.3 shows haemoglobin concentration in blood of 50
colorectal cancer patient.
Table 4.3: Haemoglobin concentration in 50 cases of colorectal cancer patients
Hb (gm/dl) N=50 Male Female
<10 13(26%) 5(38%) 8(62%)
>10 37(74%) 24(65%) 13(35%)
4.4 CEA (Carcinoembryonic antigen) level in 50 colorectal cancer cases
CEA levels were recorded in 50 cases. It ranged from 0.31 to 672 ng/ml. CEA <5ng/ml
considered normal (Aljebreen, 2007). In 25 (50%) cases CEA was below 5 ng/ml and in
25 (50%) it was more than 5 ng/ml (Table 4.3).
Table 4.4: CEA (Carcinoembryonic antigen) level in 50 colorectal cancer cases
CEA (ng/ml) N=50 Male Female
<5 25 15(30%) 10(20%)
>5 25 19(38%) 6(12%)
4.5 : Sub site distribution of colonic cancer
Distribution of cancer among 50 cases according to the site of the colon affected were as
follows: 33 (66%) case were in rectum, 6 (12%) were in the ascending colon , 3 (6%) in
hepatic flexure of colon, 2 (4%) each in caecum, descending colon, sigmoid colon and in
the transverse colon (Table 4.5). Of the total 50 cases 37 (74%) were in the left colon
(splenic flexure to rectum) and 13 (26%) were in the right colon (caecum to upto splenic
flexure). Distribution of colon cancer in 29 male cases were : 17 (34%) in rectum, 4 (8%)
in ascending colon, 2 (4%) each in transverse colon, hepatic flexure of colon and caecum
and one(2%) each in sigmoid colon and descending colon. In 21 female cases, 16
(32%)cases were in rectum, 2 (4%) cases in ascending colon, one(2%) case each in
sigmoid colon, descending colon and hepatic flexure of colon.
Table 4.5: Site distribution of colorectal cancer from distal to proximal
Variable N=50 Male Female
Rectum 33 17(34%) 16(32%)
Sigmoid colon 2 1(2%) 1(2%)
Descending colon 2 1(2%) 1(2%)
Transverse colon 2 2(4%) -
Hepatic flexure of colon 3 2(4%) 1(2%)
Ascending colon 6 4(8%) 2(4%)
Caecum 2 2(4%) -
4.6 Size and shape of the lesion
Morphology of the growth were recorded by examining the surgically resected specimen.
Maximum diameter of the growth ranged form 3 cm to 10 cm. Grossly 25 (50%) tumours
were ulcerated growth, 22 (44%) were polypoid or exophytic growth and 3(6%) were
infiltrative or annular growth. Figure 4.2 shows growth pattern of colorectal cancer
patients.
Figure 4.2 Growth pattern of colorectal cancer cases.
4.7 Histological features of colorectal cancer cases
In 50 cases of colorectal adenocarcinoma, 44(88%) were adenocarcinoma (NOS) and
6(12%) were mucinous adenocarcinoma. Of 44 cases of adenocarcinoma (NOS), 23 (46%)
were male and 21 (42%) cases were female. All 6 (12%) cases of mucinous
adenocarcinoma were male (Table 4.6).
Ulcerating
Exophytic
Infiltrative
Table 4.6: Histological diagnosis of 50 cases of colorectal cancer
Histological type N=50 Male Female
Adenocarcinoma 44 (88%) 23 21
Mucinous adenocarcinoma 6(12%) 6 -
4.7.1: Histological grading of colorectal cancer
Of 50 colorectal cancer cases, 39(78%) cases were moderately differentiated and 11(22%)
cases were poorly differentiated. In this present study no well differentiated adeno
carcinoma was observed. All the mucinous carcinoma were included in the poorly
differentiated group (Hamilton, 2000). Male predominance was observed in both
moderately differentiated and poorly differentiated cases. Among the 39 moderately
differentiated cases, 22 cases were seen in male and 17 cases in female and of 11 poorly
differentiated cases, 7 cases were seen in male and 4 in female.
Of thirty nine moderately differentiated cases, 10(20%) cases originated in right colon and
29 (58%) cases in the left colon. Of 11 poorly differentiated carcinomas, 4(8%) cases were
in the right colon and 7(14%) cases were in the left colon. Table 4.7 shows histological
grade of colorectal cancer in 50 cases with location and sex distribution.
Table 4.7: Histological grading of colorectal cancer cases with sex and location:
Tumour differentiation Number Male Female Right
colon
Left
colon
Moderately differentiated 39(78%) 22(44%) 17(34%) 10(20%) 29(58%)
Poorly differentiated 11(22%) 7(14%) 4(8%) 4(8%) 7(14%)
4.7.2 Clinical stage of cases of colorectal cancer
Staging of colonic cancer were done in all the cases. It was done on the basis of TNM
system (Turner, 2010). The maximum number of cases 22 (44%) were in stage III, 14
(28%) cases were in stage II and 13 (26%) cases in stage I and 1 (2%) case in stage IV.
Out of 22 stage III cases, 11(22%) cases were male and 11(22%) cases were female. In
14(28%) cases of stage II, 9(18%) cases were male and 5(10%) cases were female. In stage
I there was a total 13(26%) cases, 8(16%) cases were male and 5(10%) cases were female.
One (2%) stage IV case were male patient. Table 4.8 shows TNM stage in 50 colorectal
cancer cases with sex distribution and location.
Table 4.8: TNM (Tumour, Node, Metastasis) stage with sex and location
TNM stage Number of
cases
Male Female Right
colon
Left
colon
Stage I 13(26%) 8(16%) 5(10%) 2(4%) 11(22%)
Stage II 14(28% 9(18%) 5(10%) 6(12%) 8(16%)
Stage III 22(44%) 11(22%) 11(22%) 8(16%) 14(28%)
Stage IV 1(2%) 1(2%) - 1(2%) -
TNM=Tumour Node Metastasis
4.7.3 Lymph node metastasis in 48 cases of colorectal cancer
Out of 50 resected specimen, 49 cases contained lymph nodes. Lymph nodes number
ranged from four to twenty five. Histologically lymph node metastasis were present in
23(47%) cases. Among the 23 nodal metastasis cases, 17(74%) cases metastasized from
moderately differentiated primary tumour and 6(26%) cases from poorly differentiated
primary carcinoma.
Nil
Grade-1
Grade-2
Grade-3
4.7.4 : Tumour infiltrating lymphocytes (TIL) in colorectal cancer patients
Tumour infiltrating lymphocytes (TIL) were observed in all the 50 colorectal cancer cases
and catogorized into four groups, nil or absent, grade- 1, grade-2 and grade- 3. 20(40%)
cases were in grade -1, 18(36%) cases were in grade- 2, 4(8%) cases were in grade- 3 and
the rest 8(16%) cases has no tumour infiltrating lymphocytes. Figure 4.3 show percentage
of tumour infiltrating lymphocytes in 50 colorectal cancer cases.
Figure 4.3: Various grade of tumour infiltrating lymphocytes in 50 cases.
Present
Absent
4.7.5: Lymphovascular invasion in colorectal cancer patients
Lymphovascular invasion was present in 16(32%) cases and absent in 34 (68%) cases
Figure 4.4 lymphovascular invasion in CRC cases.
Figure 4.4: Lymphovascular invasion in 50 colorectal cancer cases
4.7.6: Extracellular mucin in colorectal cancer patients
Out of 50 colorectal cancer patients percentage of extracellular mucin in the tumour tissue
were recorded. 1-49% areas of extracellular mucin was present in 18(36%) cases, >50%
extracellular mucin was present in 6(12%) cases and no extracellular mucin was found in
26 (52%) cases. Figure 4.5 shows distribution of extracellular mucin in colorectal cancer
cases.
Figure 4.5 Extracellular mucin in colorectal cancer patient.
4.7.8: Signet ring cells in 50 colorectal cancer cases
Percentage of signet ring cells in the tumour tissue were recorded. 1-49% of the tumour
cells were signet ring cells in 17(34%) cases and no signet ring cells in 33 (66%) cases.
No cases having more than 50% signet ring cells was present in this series. Table-4.9
shows percentage of signet ring cells in 50 colorectal cancer cases
Table 4.9: Signet ring cells in colorectal cancer patients
Percentage of signet ring cells Number of cases (%)
Nill (%) 33(66%)
1-49% 17(34%)
>50% -
4.7.9: Circumferential margin, perinural invasion and tumour border
configuration in colorectal cancer cases
Histologically out of 50 colorectal cancer cases circumferential margin were involved in 12
(24%) cases. Of twelve involved cases 8 (67%) were stage in III and 4 (33%) were in
stage II disease. In uninvolved 38 cases, 14 (37%) cases were in stage III, 13 (34%) cases
were in stage I, 10 (26%) were in stage II and one (3%) case in stage IV.
Out of 50 colorectal cancer cases, perineural invasion was present in 15(30%) cases. Out
of these 15 cases 10 (66%) cases had stage III disease, 3 (20%) cases had stage II disease,
one (7%) case each in stage I and stage IV. Perinural invasion was absent in 35(70%)
cases. In which 12(24%) cases each were in stage I and stage III and 11(22%) cases in
stage I disease.
In 50 cases, tumour border configurations were examined microscopically. Infiltrative
pattern were seen in 26 (52%) cases and pushing pattern in 24(48%) cases. In 26(52%)
infiltrative pattern cases, 15 (58%) cases were in stage III, 6 (23%) cases in stage II, 4
(15%) cases in stage I and one (4%) case in stage IV. In 24 cases having pushing border, 9
(37%) cases were in stage I, 8 (33%) cases in stage II, 7 (33%) cases in stage IV. Table
4.10 shows Circumferential margin involvement, Perineural invasion and Tumour border
configuration with TNM stage
Table 4.10: Circumferential margin involvement, Perineural invasion and
Tumour border configuration with TNM stage.
TNM
stage
Circumferential margin Perinural invasion Tumour border
configuration
Involved
N=12
Not
involved
N=38
Present
N=15
Absent
N=35
Infiltrative
N=26
Pushing
N=24
Stage-1 - 13(34%) 1(7%) 12(34%) 4(15%) 9(37%)
Stage-2 - 10(26%) 3(20%) 11(32%) 6(23%) 8(33%)
Stage-3 4(33%) 14(37%) 10(66%) 12(34%) 15(58%) 7(30%)
Stage-4 8(67%) 1(3%) 1(7%) - 1(4%) -
4.8 DNA Methylation in 24 colorectal cancer tissue and
corresponding normal colonic tissue
Genome-wide methylation status was interrogated at nucleotide resulotion in 27500 CpG
loci. In figure 4.6 scatter diagram shows a linear correlation between the total intensity
(methylated + unmethylated) of tumour and normal tissue. Strong positive correlation
was seen which indicates there is no bias in the microarray intensity data. Figure 4.6
shows total intensity of ethylation data in all 24 cases.
Figure 4.6: Scatter diagram showing total intensity in tumour tissue and its
corresponding normal tissue in all 24 cases
4.8.1 Methylation changes in tumour and surrounding normal tissue
There was significant difference in the methylation status between tumour and sorrounding
normal tissue.
4.8.1.1 The average beta
Average beta is the methylation status of a gene locus. It is calculated by intensity of the
methylated sequence(x)/ [intensity of the methylated sequence (x)+ intensity of the
unethylated sequence (y) ]. The ratio is between 0 to 1, where 0= extremely
hypomethylated and 1= extremely hypermethylated. Figure 4.2 shows scatter plot of
average beta for tumour tissue against that of corresponding normal tissue in 24 cases. The
figure shows that in the tumor tissue, there are a number of loci that are hypomethylated
compared to the corresponding normal tissue and a number of loci that are
hypermethylated. The central line represents regression line and other two lines on either
side represents the boundary for two fold change
Fig 4.7: Scatter diagram showing average beta of all 48 samples
4.8.1.2 Tumour differential score
Tumour differential score indicates how significant the differenceis between the
methylation status of the normal and tumour tissue. The more the positive value, the more
that is hypermethylation. The more negative the more hypomethylation. Usually more
than 30 is significant and indicates hypermethylation. Figure 4.4 shows some cases
beyond +30 which are hypermethylated loci and many data beyond -30 which indicates
hypomethylated loci. It also shows majority of the data clustering around 0 which
indicates no differential methylation pattern in many of the cases.
Figure 4.8: Histogram showing tumour differential score.
4.8.2 Factors responsible for the change in methylation data
Bar diagram ( Fig 4.6) shows the significance of the different sources of variation in the
total methylation data done by ANOVA test. It indicates the factors that influence the
variation of the methylation data in 48 samples. It shows significance of factors
responsible for the methylation changes in tumour tissue and normal tissue . Difference of
the tissue (whether tumour or normal), sex (male or female, case id i.e case to case
variation and location of the lesion (proximal or distal colon).
Fig 4.9: Bar diagram showing sources of variation of methylation data.
Another important factor that is responsible for the changes in the methylation is tumour
differentiation. In this study in 24 cases 6 cases were poorly differentiated tumour and rest
were moderately differentiated tumour. There were changes in methylation pattern due to
differentiation (Figure 4. 7).
Figure 4.10 Variation of methylation data due to tumour differentiation.
4.8.3 Genes involved in the methylation difference in colonic tumour
tissue and normal colonic tissue
Some of the candidate genes are identified in this study which show methylation difference
in case of tumour tissue compared to normal colonic tissue. But which of these hypo or
hypermethylated genes play role in CRC and what are their exact role are yet to be
determined and needs further study. Table 4.11 show some genes that are methylated in
colorectal cancer in this study.
Table 4.11: Selected genes that show methylation differences in 24 cases of
Colorectal carcinoma.
Genes Tumour AVG. Beta Normal AVG Beta DiffScore Delta Beta
EYA4 0.516264 0.149085 362.88 0.367179
TFPI2 0.422315 0.100107 362.88 0.322208
GATA4 0.569414 0.243687 362.88 0.325726
CDKN2A 0.422516 0.159246 362.88 0.263270
DCC 0.566206 0.289243 362.88 0.276963
MDF1 0.550374 0.083746 362.88 0.461493
ITGA4 0.630950 0.122244 362.88 0.508707
GAD2 0.430238 0.155448 362.88 0.274790
ADCY4 0.474352 0.151537 362.88 0.322815
CCNA1 0.663816 0.300486 362.88 0.363330
DAB21P 0.455048 0.036997 362.88 0.41805
5.0 DISCUSSION
In this study, 50 cases of colorectal cancer were analyzed to find out histomorphological
features, selected clinical data and DNA methylation changes in 24 cases.
The mean age of the 50 cases was 47±14.8 years. The age range was from 19-84 years
with male and female ratio of 1.4:1. 58% of the cases were below the age of 50 years.
Peak incidence of colorectal cancer in this study were 50-59 years which is lower than that
of western and other countries (Table 5.1). Turner (2010) found only 20% of the cases
below 50 years. Keating et al (2003) found only 6.3% cases below 50 years. (Table-5.1).
Table 5.1 Age and sex distribution of CRC in different studies and the present
study.
Investigator/ Country
Peak
incidence
(Years)
Mean age
(Years)
Range of
age (Years)
Cases <50
years
Male :
female
Turner (2010),USA 60-70 <20% 1.2:1
Gomez et al (2004), UK 60-70 70±11 01-90 1.5 1
Riddel et al (2003), USA 60-70 60 3 2
Keating et al (2003),
New Zealand
70-80 69 6.3 Equal
Hossain(2007)
Bangladesh
40-49 44.1±16.2 11-75 53% 1.6:1
Present study 50-59 46.6±14.8 19-84 58% 1.4:1
54
The mean age of colorectal cancer in this present study indicate that colorectal carcinoma
is relatively common in lower age group in our country. Though incidence of colorectal
cancer in Bangladesh is not exactly known, it appears to be common in younger age group.
This may be due to both environmental factors and genetic factors. Leon et al (2004)
commented on recent trends in colorectal cancer. They observed rapid increase of CRC in
the developing countries. Gomez et al (2004) commented a tendency of right sided shift
from left sided colorectal cancer distribution. Keating et al (2003) suggested right sided
cancer to be less well differentiated than the left sided CRC. Presently we do not know
whether the CRC changing trends in Bangladeshi population i.e early age of onset, Right
colon shift etc.
It was also observed in present study that there is slight male preponderance regarding
colorectal cancer cases. Keating et al (2003) found equal gender distribution in their study.
Rectal bleeding was the commonest presentation (40% cases) at the time of first
consultation. The other presenting complaints were abdominal pain (16% cases), altered
bowel habit (12% cases), anorexia, pallor and generalized weakness (12% cases). Palpable
abdominal mass, partial intestinal obstruction and weight loss were present in addition to
the major complaints. Though abdominal pain has been described by some to be the
commonest symptom in colorectal cancer (Aljebreen, 2007), it was not the case in this
present study. Only 8(16%) cases had this feature. One possible explanation is negligence
on the part of the patient and frequent use of analgesics.
In this study, 20(40%) patients presented with per rectal bleeding, is similar to other study
(Hossain, 2007). Per rectal bleeding was observed in 17(85%) cases of left colon cancer.
55
Most of the cases with per rectal bleeding, tumour was present in rectum which explains
partly that per rectal bleeding may be most common symptoms in rectal cancer.
Anaemia was present in 13(26%) cases. Among these anaemic patients with colorectal
cancer 8 cases were female and 5 cases were male. About 69% of cases with cancers in
the right sided colon were anaemic and 31% of the patients with left sided cancer had
anaemia. Ayyub et al(2002) observed anaemia in 55% of their cases and also mentioned
70% cases were in right sided colon and 30% cases in left sided colon. This present study
shows lower haemoglobin levels for right sided colon cancer.
CEA (Carcinoembryonic antigen) level in serum were ranged from 0.39 ng/ml to 672
ng/ml with mean level 26.6. CEA was elevated (>5 ng/ml) in 25(50%) cases. Rosai
(2004) commented CEA more than 5 ng/ml is the adverse prognosis factor in CRC. In this
present study, in 25 cases having CEA more than 5 ng/ml, 17(68%) cases were in stage III
and one case was in stage I. This shows majority of the patients having elevated CEA are
in high stage disease. Aljebreen (2007) found 32% of cases of colorectal cancer with
elevated CEA level. Ayyub et all (2002) found CEA level ranged from 1 to 850.2 ng/ml
with a mean of 48.62.
The sub site distribution of colorectal cancer in this study shows 37(74%) cases were in the
left colon and 13(26%) cases in the right colon. Recent trend of shifting of CRC towards
right colon observed by Gomez et al (2004) is not supported by this present study.
However it is similar to the studies done by other researchers. This is shown in table 5.2.
56
Table-5.2: Distribution of colorectal cancer in left colon and right colon.
Investigator Left colon Right colon
Riddell et al(2003), USA 75% 25%
Leon et al (2004), Italy 70% 30%
Ayyub et al (2002), Saudi Arabia 70% 30%
Hossain(2007), Bangladesh 63% 37%
Present study 74% 26%
In Among 37 cases of left colon cancer cases, 33 cases were in rectum. This shows higher
percentage of rectal cancer in this study.
Size and shape of the tumour were recorded in all 50 cases. The size of the tumour ranged
from 3 cm to 10 cm. Pattern of growth were, 25(50%) cases ulcerating, 22(44%) cases
exophytic/polypoid and the rest 3(6%) infiltrative. 18(72%) of the ulcerated lesion was
present in rectum. This may be one of the probable cause of per rectal bleeding in most of
the rectal cancer cases.
On histological examination of 50 colorectal cancer cases, 44(88%) of cases were usual
adenocarcinoma and 6(12%) cases were mucin secreting adenocarcinoma. Among these
39(78%) cases were moderately differentiated and 11(22%) cases were poorly
differentiated cancer. No well differentiated cancer was reported in this series. Ekem et al
(2008) found 37% moderately differentiated, 17% well differentiated and 6% poorly
differentiated cancer. Keating et al (2003) found right sided tumour were less well
differentiated than left sided tumour. In this study. of thirty nine moderately differentiated
cases, 10(26%) originated in right colon and 29 (74%) in the left colon. Of the 11 poorly
57
differentiated carcinomas, 4 were in the right colon and 7 were in the left colon. In this
study left colon cancer were less well differentiated than right side.
Tumour infiltrating lymphocytes were observed in all 50 cases. 20(40%) cases were in
grade 1, 18(36%) cases were in grade 2, 4(8%) cases were in grade 3 and the rest 8(16%)
cases has no tumour infiltrating lymphocytes. In grade III TIL cases, only one were in
stage III compared to 2 in stage I. In no TIL cases, 4 were in stage III disease compared to
2 in Stage I disease. It shows higher TIL is associated with lower stage though conclusion
can not be made from this small sample size.
Lymphovascular invasion was present in 16(32%) cases and absent in 34 (68%) cases.
Ekem et al (2008) found lymphovascular invasion in 33% cases. Out of 16 positive cases
12(75%%) cases had stage III disease and 3(19%) cases had stage II and one (6%) case in
stage IV disease, which indicates higher stage disease in lymphovascular invasion positive
tumour. As stage is the major factor in predicting prognosis (Rosai, 2004). In this study,
majority of the cases in stage III disease show lymphovascular invasion.
This partly explains the importance of reporting lymphovascular invasion and Tumour
infiltrating lymphocytes in surgical pathology report.
Percentage of signet ring cells in the tumour tissue were recorded. 17(34%) cases show 1-
49% signet ring cells and 33 (66%) cases with no signet ring cells. No case having more
than 50% signet ring cells was present in this series.
58
Out of 50 colorectal cancer cases circumferential margin were involved in 12 (24%) of
the cases. Of twelve involved cases 8 cases were in stage III and 4 cases were in stage II
disease. Perineural invasion was present in 15(30%) cases. Out of 15 cases 10 cases had
stage III disease, 3 cases had stage II disease and one case each in stage I and stage IV.
Tumour border configuration were examined microscopically. Infiltrative pattern was seen
in 26 (52%) cases and pushing pattern were seen in 24(48%) cases. In 26 infiltrative
pattern cases, 15 cases in stage III, 6 cases in stage II, 4 cases in stage I and one case in
stage IV. This study shows higher stage disease in circumferential margin involved,
perinural invasive and tumour with infiltrative border cases. Rosai (2004) commented
circumferential margin, perineural invasion and infiltrative tumour border are adverse
prognostic factors in colorectal cancer (CRC).
TNM Staging (Turner, 2010) of colorectal cancer was done in all the cases. The
maximum number of cases 22 (44%) were in stage III, 14 (28%) were in stage II and 13
(26%) in stage I and one (2%) in stage IV. This study shows only 26% stage 1 cases
which indicates delay in diagnosis of the colorectal cancer cases. Derwinger et al (2010)
found 41% cases in stage III, 36% cases in stage II, 12% cases in stage IV and 10% cases
in stage I.
In this present study 49 colon cancer specimens were accompanied by lymph nodes. Of
these 23(47%) had nodal metastasis. It indicates patients are in advanced stage at the time
of attending physician. In 23 nodal metastasis cases 17(74%) were moderately
differentiated and 6(26%) cases were poorly differentiated tumour.
59
In addition to environmental factors, genetic and epigenetic mechanisms (DNA
methylation) are involved in the pathogenesis of Colorectal carcinoma (Turner, 2010).
DNA methylation abnormalities is such important epigenetic changes in CRC.
Methylation abnormalities are also observed in other tumours like stomach , pancrease,
breast, lung cancers (Samarakoon, 2010).
In this study DNA methylation changes was anlyzed in 24 cases of CRC tumour tissue and
corresponding normal colonic tissue. This study shows methylation difference between
the tumour and normal colonic tissue. A number gene loci are hypermethylated compared
to normal tissue and a number of gene loci are hypomethylated compared to normal tissue
(Figure 4.8).
This study also shows the factors responsible for methylation changes in tumour tissue.
Methylation difference is observed between individual cases, location of tumour (right
colon tumour tissue is compared with left colon tumour tissue) and between male and
female tumour tissue. Methylation difference was also observed between poorly
differentiated and moderately differentiated adenocarcinoma (Figure 4.10).
Some of the candidate genes showing hypo or hypermethylation is also identified and
shown in table 4.11 in result observation section. Kim et al (2010) also found in their
study that GATA4 gene is 70% methylated in CRC compared to only 6% methylated in
normal colonic tissue and ITGA4 gene is 92% methylated in CRC compared to 13%
methylated in normal tissue.
60
The exact role of DNA methylation in molecular pathogenesis of cancer is not fully
understood. It needs further study to conclue the role of DNA methylation in the
pathogenesis of colorectal carcinoma.
61
6.0 Summary and conclusion
6.1 Summary
This study was carried out to find histomorphological features, selected clinical data in 50
colonic cancer cases and DNA methylation changes in 24 of these cases. In this study the
mean age was 47±14.8 years and 58% of the cases were below the age 50 years which was
10 years less than that described by western observers. Male cases appears to be slightly
more than females.
Clinical features and related investigations are more or less similar that of other countries.
Recent trend show right shift (more cancers in the right side than left sided colon) of CRC
in western countries but is is not the findings in this study.
Adenocarcinoma of usual pattern was most common type, other common type was mucin
secreting adenocarcinoma. 78% cases were moderately differentiated and 22% cases were
poorly differentiated. No well differentiated cases were observed in this series indicates
higher grade tumours in this study subjects. Most common stage was stage III (44%) cases
and 24% cases were in stage I. Lymphnode metastasis was present in 47% cases which
indicates delay in diagnosis and advanced stage of disease.
DNA methylation changes was seen in tumour tissue compared to normal tissue. Other
factors for methylation changes were location of the tumour, differentiation of the tumour
and case to case variation. Some genes were identified which are methylated in colorectal
cancer tissue but needs further study with large sample size to specify the association.
6.2 Conclusion
Histomorphological and selected clinical data presented in this study are more or less
similar to that in other published reports, with the exception of mean age of the cases
which is about one decade less than western population. DNA methylation changes are
observed in the tumour tissue. Some genes are also identified but needs further study to
confirm the association and their role in pathogenesis.
7.0 BIBLIOGRAPHY
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Acknowledgements
I convey my respect and gratitude to my teacher honorable professor Dr.
Mohammed Kamal, Chairman, Department of Pathology, Bangabandhu Sheikh
Mujib Medical University (BSMMU), Dhaka and supervisor of this study for
his guidance, valuable suggestions, constant supervision, affectionate advice
and whole hearted co-operation in making this study a success. Without his
vigilance and care this research would have been difficult.
I would like to express my sincere gratitude to my teacher Professor (Dr.)
Ashim Ranjan Barua for his valuable suggestions and co-operation.
I would pay my deep gratitude to my teacher Professor (Dr.) Kamrul Hasan
Khan for his valuable advice and co-operation.
I am thankful to my teacher Dr. Tamanna Choudhury, Associate professor,
Department of Pathology, BSMMU, Dhaka for her valuable suggestions and
co-operation.
I am greatly thankful to Professor Pran Gopal Datta, Vice-Chancellor, BSMMU, Dhaka for
providing me an opportunity to perform this study in this University.
I am grateful to my teacher and co-supervisor, Dr. Ferdousy Begum, Assistant
Professor, Department of Pathology, BSMMU, for her advice, meticulour
correction and active participation in this study.
I am highly grateful to my teachers Dr. AKM Nurul Kabir, Dr. Sultana
Gulshana Banu, Dr. Suraiya Enam, Dr. Saequa Habib and Dr. Shabnom
Akhter, Assistant Professors, Department of Pathology, BSMMU for their help
and co-operation.
I must thank and pay my gratitudes to DR. Muhammad G. Kibriya and Dr. Farzana
Jasmine, Research associate- Assistant Professors, Department of Health Studies (BSD),
The University of Chicago Medical Center, Chicago , IL , USA, for their constant
encouragement, support, suggestions and immediate response to my e-mails regarding this
study.
I am very grateful to Prof. Zahidul Haq, Professor, Department of Surgery, BSMMU for
his help, co-operation and suggestions for collecting specimen for this study.
I would like to extend my sincere thanks to all my colleagues specially Dr. Himel, Dr.
Tahera, Dr. Nizam, Dr. Babul, Dr. Taufiq, Dr. Rupash, Dr. Alamgir, Dr. Shahadat and all
other residents for their advice, sympathy and co-operation during my study.
I would like to appreciate Mr. Osman Goni Chowdhury, Mr. Ruhi Das Roy and other staff
of the Department of Pathology, BSMMU for their help and co-operation.
I am thankful to Mr. Shelabrata Barua (Jewel) and Md. Faruk hossain who took the trouble
to computer compose and typing this thesis.
I acknowledge all patients involved in this study and their relatives for co-operation and
providing me valuable informations that were helpful in the research work.
I wish to express my highest regards and heartiest gratitude to my beloved parents Md.
Muzibur Rahman Sarkar and Mrs. Khaleda Begum who always wanted me to be a good
and honest teacher. My two younger brothers who always supported me and help me to
take any desision. And also my students who inspired me to be a good teacher and
researcher.
All credit belongs to almighty Allah.
Septeber, 2010 Dr. Abu Khalid Muhammad Maruf Raza
Thesis book
APPENDIX-II
Clinical proforma
Title: Study on pathological aspects of colorectal carcinoma.
Identification:
1.Case number-
2.Lab number-
3.Date-
4.Name-
5.Age-
6.Sex-
7.Adress-
8.Occupation-
Presenting complaints:
Tumor Location:
Family history:
Socio-economic history:
Relevent investigation: Haemoglobin-
ESR-
CEA-
Others-
Gross examination: Specimen type-
Tumor type-
Tumor configuration-
Tumor size-
Mesorectum-
Microscopic examination:
ii
Histologic type-
Histologic grade-
Extent of invasion-
Margins- Proximal-
Distal-
Circumferential-
Lymphovascular invasion-
Perinural invasion-
Tumour infiltrating lymphocytes-
Tumor border configuration-
Signet ring cells-
Extracellular mucin-
Additional pathologic findings-
Diagnosis:
Consultant pathologist:
iii
APPENDIX -III
PREPARATION OF STAINS AND CHEMICALS:
HAEMATOXYLIN AND EOSIN STAINS:
a) Harris’ Haematoxylin:
Haematoxylin crystal 5.00 gm
Absolute alcohol 50.ml
Ammonium alum 100.00 gm
Distilled water 950. ml
Mercuric oxide 2.50 gm
Glacial acetic acid 40.00 ml
Procedure: Haematoxylin crystals were dissolved in alcohol at 56 C in an oven. Alum
was also dissolved by heating. These two solutions were mixed thoroughly and brought to
boil rapidly. Then it was removed from the flame and mercuric oxide was added slowly.
The solution was then allowed to cool rapidly in cold water. The solution was ready to use
after cooling. It was kept in room temperature and filtered before use. 2-4 ml of glacial
acetic acid was added per 100 ml of solution to enhance the precision of nuclear stain.
b) Acid alcohol:
Hydrochloric acid (pure) 1 ml
70% ethyl alcohol (mixed thoroughly) 99 ml
c) Eosin solution:
Eosin (water soluble) 1gm
Distilled water 100 ml
Procedure : Eosin dissolved in distilled water and mixed thoroughly. It was stored at room
temperature and filtered before use. Solution was made more intense by adding glacial
acetic acid in the proportion of 0.2 ml for 100 ml solution before use.
iv
APPENDIX-IV
TISSUE PROCESSING AND STAINING PROCEDURE
Tissue processing was done in Department of Pathology, BSMMU by standard protocol in
automatic tissue processor (BAVIMED 2050, BAVIMED Laborgeneratebau GmBH,
Birkeau, GERMANY).
Processed tissue than properly embedded on melted paraffin for making blocks and
sections. The sections were stained with haematoxylin and eosin for microscopic
examination.
Paraffin embedding: Metallic moulds were used for this purpose. These moulds were first
lubricated with liquid paraffin. Then melted paraffin was poured into it. The tissue was
carefully embedded in proper plane at the bottom of the mould. The respective number of
the tissue was inserted into the paraffin wax by the side of the mould. The melted paraffin
was then allowed to harden at room temperature. After hardening, the mould was removed
and blocks were trimmed properly to mount on a block holder. Then the blocks were kept
in ice chamber of a refrigerator for sometime before cutting the sections.
Section cutting: Each block of tissue mounted on the holder was fitted in the microtome
machine. The microtome knife was properly sharpened before. A water bath with regulated
temperature of 45 to 50 C was used for floatation. Sections were cut at 4-5 micron
thickness. Ribbons of good sections were selected and floated on luke warm water in the
water bath. The sections were then taken on albuminized glass slides. The slides were kept
in inclined position to drain off excess water and allowed to dry at room temperature.
Staining: All the slides for histopathological examination were stained by routine
Haematoxylin and Eosin method
v
SPTEPS OF HAEMATOXYLIN AND EOSIN STAINING:
a) Deparaffinization :
i) Slides were kept in hot air oven 15 minutes
ii) Xylol- I 5 minutes
iii) Xylol –II 5 minutes
b) Hydration :
i) Absolute alcohol – I 3 minutes
ii) Absolute alcohol –II 2 minutes
iii) 95% alcohol 2 minutes
iv) 70% alcohol 2 minutes
v) 50% alcohol 2 minutes
vi) Running tap water 2 minutes
c) Staining with Haematoxylin and Eosin:
Harris’s Haematoxylin 10 minutes
1% acid alcohol 1 minutes
Section were placed in running tap water till the sections become blue. Counter staining
was done by immersing the section in 1% watery solutions of eosin for 1 minute.
d) Dehydration:
The sections after Haematoxylin and Eosin staining were dehydrated in
following manner.
i) 50% alcohol 02 minutes
ii) 70% alcohol 02 minutes
iii) 80% alcohole 02 minutes
iv) 95% alcohol 02 minutes
v) absolute alcohol I 02 minute
vi) absolute alcohol II 02 minutes
vi
e) Clearing:
a) Xylol – I - 5 minutes
b) Xylol – II - 5 minutes
c) Xylol – III - 2 minutes.
6) Mounting:
Mounting was done with DPX using No. 1 cover slip.
Results:
Cell nuclei - Blue
Cytoplasm - Pink
Collagen fibre - Pink
RBC - Bright red
vii
APPENDIX-V
Protocol for DNA extraction and purification from colonic tissue (Gentra Puregene
Handbook 04/2010, QIAGEN company, USA (www.qiagen.com). :
1. Dissect tissue sample quickly and freeze in liquid nitrogen. Grind frozen tissue in liquid
nitrogen with a mortar and pestle. Work quickly and keep tissue on ice at all times,
including when tissue is being weighed.
2. Dispense 300 tube on ice, and add the
ground tissue from the previous step. Complete cell lysis by following step 2a or 2b below:
2 a. Heat at 65°C for 15 min to 1 h.
2 b. If maximum yield is required, add ene Proteinase K, mix by inverting
25 times, and incubate at 55°C for 3 h or until tissue has completely lysed. Invert tube
periodically during the incubation. The sample can be incubated at 55°C overnight for
maximum yields.
n, and mix the sample by inverting 25 times. Incubate at
37°C for 15–60 min.
4. Incubate for 1 min on ice to quickly cool the sample.
20 s at high speed.
6. Centrifuge for 3 min at 13,000–16,000 x. The precipitated proteins should form a tight
pellet. If the protein pellet is not tight, incubate on ice for 5 min and repeat the
centrifugation.
and add the
supernatant from the previous step by pouring carefully. Be sure the protein pellet is not
dislodged during pouring.
Glycogen Solution
8. Mix by inverting gently 50 times.
9. Centrifuge for 1 min at 13,000–16,000 x g.
10. Carefully discard the supernatant, and drain the tube by inverting on a clean piece of
absorbent paper, taking care that the pellet remains in the tube.
pellet.
12. Centrifuge for 1 min at 13,000–16,000 x.
13. Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper,
taking care that the pellet remains in the tube. Allow to air dry for 5 min.
The pellet might be loose and easily dislodged. Avoid over-drying the DNA pellet, as the
DNA will be difficult to dissolve.
speed to mix.
15. Incubate at 65°C for 1 h to dissolve the DNA.
16. Incubate at room temperature overnight with gentle shaking. Ensure tube cap is tightly
closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a
storage tube.
APPENDIX-VI
Protocol DNA methylation (Infinium Assay Methylation Protocol Guide, Catalog # WG-
901-2701 Part # 11322371 Rev. A,Illumina Inc, USA.)
1. Bisulphite conversion of DNA material:
After DNA extraction bisulphite conversion of DNA was done by using the EZ DNA
MethylationTM
Kit (Zymo Research, Catalogue # 500, USA). The kit is based on the reaction
that takes place between unconverted cytosine and sodium bisulfite where cytosine is converted
into uracil. With subsequent amplification this converted Uracil becomes T (thimine). So instesd
of unmethylated C we get T in the sequence. 1 µg DNA from each tissue sample were used for
bisulfite conversion. These bisulphite converted DNA were used immediately for methylation
assay.
2. Amplification of DNA:
The Bisulfite converted DNA samples were denatured by alkali to open up the strands and then
was isothermally amplified using Multi-sample Amplification. Master Mix provided by Illumina
by overnight incubation.
3. Fragmentation, precipitation and hybridization of DNA:
The amplified product was fragmented by a controlled enzymatic process (FMS reagent provided
by Illumina. The process uses end-point fragmentation to avoid overfragmenting the samples.
These fragmented DNA was precipitated by adding and spinning with isopropanol. Then the
x
precipitated DNA was resuspended in hybridization buffer (RA1 reagent provided by Illumina).
After resuspension, the fragmented DNA was heat denatured. Then samples were put on
methylation bead chip. Twelve samples are applied on to each BeadChip, which keeps them
separate with an IntelliHyb seal. The prepared BeadChip is incubated overnight in the Illumina
Hybridization Oven at 48° c.
4. Wahing, extension and staining of Beadchips:
On the following day unhybridized and non-specifically hybridized DNA were removed by
washing, and the chips were prepared for staining. A single base extension reaction of the
hybridized methylated or unmethylated sequence was done by incorporating fluor tagged
necleotide.
5. Imaging Bead Chip:
This was done by using Illumina Bead Array Reader software. Intensity of methylation was
calculated by intensity of the methylated sequence(x)/ intensity of the methylated sequence (x) +
intensity of the umethylated sequence (y). If both the alleles are methylated, intensity = x/x+0=1
and if both the alleles are unmethylated then intensity = 0/0+y=0 and if one allele is methylated
and the other is unmethylated then intensity is=x/x+y.
xi
Appendix - VII
American Joint Committee on Cancer (AJCC) TNM Classification of
Colorectal Carcinoma (Turner, 2010).
TUMOUR
Tis In situ dysplasia or intramucosal carcinoma
T1 Tumor invades submucosa
T2 Tumor invades into, but not through, muscularis propria
T3 Tumor invades through muscularis propria
T3a Invasion <0.1 cm beyond muscularis propria
T3b Invasion 0.1 to 0.5 cm beyond muscularis propria
T3c Invasion >0.5 to 1.5 cm beyond muscularis propria
T3d Invasion >1.5 cm beyond muscularis propria
T4 Tumor invades adjacent organs or visceral peritoneum
T4a Invasion into other organs or structures
T4b Invasion into visceral peritoneum
REGIONAL LYMPH NODES
NX Lymph nodes cannot be assessed
N0 No regional lymph node metastasis
N1 Metastasis in one to three regional lymph nodes
N2 Metastasis in four or more regional lymph nodes
DISTANT METASTASIS
MX Distant metastasis cannot be assessed
M0 No distant metastasis
M1 Distant metastasis or seeding of abdominal organs
TNM staging for colorectal carcinoma (Turner, 2010)
Stage-I Tl N0 M0 or T2, N0, M0
Stage-II T3 N0 M0 or T4, N0, M0
Stage-III T any Nl M0 or T any,N2,N3, M0
Stage-IV T any N any Ml
Appendix–VIII
MasterTable
Sl.No.
CaseID
Lab.No.
Age/Sex
C/F
Hb(mg/dl)
CEA
(ng/ml)
Location
Size(Cm)
Diagnosis
Differentia
tion
TILgrade
Ext.Muc
Signetring
cell
LV
invasion
PN
invasion
Tumour
border
Circumfer
ential
margin
LN/Inv
Growth
pattern
Tumour
stage
1C1B-7750/0935/Fp/r/b11.18.01Rec6AdeM2NoNoPrePInfInv11/11UlIII
2C2B-114-16/1019/Mp/r/b10.83.14Rec4AdeMNNoNoAPInfInv7/0ExoII
3C3KA-119-2375/Mp/r/b11672Rec3AdeMNNoNoAAPushInv7/3UlIII
4C4B-174/1068/Mpain1110.9Cae10MucP1>50%1-49%AAPushFree10/0ExoII
5C5B-107-8/1041/Mp/r/b
and
pain
73.9Tran6MucPN>50%1-49%AAPushFree4/0ExoI
6C6B-268/1052/F/p/r/b9.316.8Rec4AdeP1NoNoAAPushFree8/0ExoI
7C7B-273/1055/Mp/r/b11.279.64Rec6MucPN>50%1-49%AAPushInv0/0InfII
8C8B-327/1025/Mp/r/b10.83.9Rec4AdeM1NoNoPrePInfFree7/4ExoIII
9C9B-331/1052/Mpain104.1Asc6AdeM11-49%1-49%PreAInfFree11/1UlcIII
10C10KA-266-6965/Fa/b11.19.3Rec8AdeM11-49%NoAAInfFree11/5ExoIII
11C12B-444/1045/Mp/r/b1318.34Rec5AdeM11-49%NoPreAInfFree4/2UlcIII
12C13KA-399-40245/Mp/r/b9.39.1Rec7AdeM1NoNoAAInfFree4/0UlcII
13C14KA-406-0743/Mp/r/b10.111.1Rec5AdeMNNoNoAAInfFree7/0ExoI
14C15KA423-26-35/FPain1168.9Rec5AdeP11-49%1-49%AAInfFree12/1UlcIII
15C16B-566/1035/Mp/r/b1113.7Rec6AdeM1NoNoPrePInfiltr
ative
Free7/1ExoIII
xiv
Appendix–VIII
MasterTable
Sl.No.
CaseID
Lab.No.
Age/Sex
C/F
Hb(mg/dl)
CEA
(ng/ml)
Location
Size(Cm)
Diagnosis
Differentia
tion
TILgrade
Ext.Muc
Signetring
cell
LV
invasion
PN
invasion
Tumour
border
Circumfer
ential
margin
LN/Inv
Growth
pattern
Tumour
stage
16C17B-589/1024/MPain12.7
0
0.31Cae10MucP1>50%NoAAPushInv20/15UlcIII
17C19B-960/1059/Mp/r/b10.61.9Hepa6AdeP2NoNoAAPushFree20/0ExoII
18C20B-1126/1028/Ma/b10.63.1Rec9AdeM1NoNoAAPushFree14/0ExoI
19C21B-1370/1053/Mp/r/b10.224.6Rec5AdeM21-49%NoAAInfFree7/0ExoI
20C22B-1433/1084/Fmass11.64.44Rec4AdeM11-49%NoAAPushFree11/0UlcI
21C23B-1467/1030/Ma/b123.9Rec7AdeM1No1-49%PrePInfFree19/1UlcIII
22C25B-1706/1027/Fp/r/b
and
pain
11.13.1Hepa6AdeM11-49%NoprePInfFree23/1UlcIII
23C26KB-705-0848/Fp/r/b123.19Rec4AdeM3NoNoAAPushFree10/0ExoI
24C27B-1851/1036/Mp/r/b93.9Rec10AdeM1NoNoAAInfFree12/0ExoI
25C28B-1995/1028/Fp/r/b13.13.9Rec4AdeM31-49%1-49%preAInfFree4/0UlcII
26C30KC-61-
63/10
59/Fpain11.841.2Rec7AdeM31-49%NoAAInfFree8/0ExoI
27C31BH-779-
80/10
50/Mp/r/b
and
pain
11.63.9Asc6AdeM21-49%NoAAInfFree4/0ExoII
28C32KC-320-2342/Ma/b131.81Rec3AdeM11-49%1-49%AAPushFree2/0ExoI
29C33B-2397-
99/10
58/Fa/b12.85.5Rec6AdeM1NoNoPrePInfFree15/0ExoII
30C34B-2415-
16/10
37/Mp/r/b14.5314Des5MucPN>50%1-49%PrePInfInvolve
d
17/17InfIII
xv
Appendix–VIII
MasterTable
Sl.No.
CaseID
Lab.No.
Age/Sex
C/F
Hb(mg/dl)
CEA
(ng/ml)
Location
Size(Cm)
Diagnosis
Differentia
tion
TILgrade
Ext.Muc
Signetring
cell
LV
invasion
PN
invasion
Tumour
border
Circumfer
ential
margin
LN/Inv
Growth
pattern
Tumour
stage
31C35B-2626/1045/Fa/b11.27.49Rec8AdePN1-49%1-49%AAInfFree15/1ExoIII
32C36B-2613/1045/Fa/b12234Des4AdeMNNoNoPrePInfInv25/20UlcIII
33C37KD-236-4140/Fw/p13.13.38Rec5AdeM2No1-49%PreAPushFree9/3UlcIII
34C38KD236-41-73/Mp/r/b
and
pain
13.524.8Hepa3AdeM2
1-49%1-49%
AAPushFree15/0UlcI
35C39B-2888/1050/Fpain5.42.17Sig5AdeP2No1-49%APPushFree16/0UlcII
36C40KD-715-1966/Mp/r/b14.62.1Rec4AdeM1NoNoAPPushFree10/0UlcI
37C41KD-809-1130/Fp/r/b
and
pain
12.74.1Rec7AdeM2NoNoAAPushFree8/0ExoI
38C42B-2976/1050/Fw/p10.93.75Rec5AdeM21-49%NoAAPushi
ng
Free7/2UlcIII
39C43B-3376-
79/10
28/Mp/r/b
and
pain
13.813.57Rec5.5MucP1>50%1-49%AAInfInv5/2UlcIII
40C44B-3374/1052/Mmass8.84.34Rec5AdeM1NoNoAAPushFree15/9UlcIII
41C45KD-868-7150/Ma/b6.91.63Tran4.5AdeM3NoNoAAPushFree4/3ExoIII
42C46B-3395-9663/Fp/r/b12.738.9Rec5AdeM2NoNoAAPushFree5/0UlcII
43C47B-3469/1038/Fa/b1029.3Asc8AdeM2NoNoAPInfFree7/0UlcIII
44C48B-3484-
85/10
50/Mw/p11.6256Asc3AdeM21-49%NoPrePInfFree7/4UlcIV
45C49B-3604-
05/10
48/Fw/p10.75.1Rec4AdeM21-49%NoAAPushFree6/0ExoII
xvi
Appendix–VIII
MasterTable
Sl.No.
CaseID
Lab.No.
Age/Sex
C/F
Hb(mg/dl)
CEA
(ng/ml)
Location
Size(Cm)
Diagnosis
Differentia
tion
TILgrade
Ext.Muc
Signetring
cell
LV
invasion
PN
invasion
Tumour
border
Circumfer
ential
margin
LN/Inv
Growth
pattern
Tumour
stage
46C50B-3738-
39/10
65/Mw/p815.9Asc10AdeM21-49%1-49%AApushInv11/0ExoII
47C51B-3906/1039/Fw/p8.61.9Rec6AdeP21-49%1-49%PrePInfInv9/9InfIII
48C52B-4307/1039/Fmass8.11.09Sig5AdeM2NoNoPreAInfFree7/0UlcII
49C53B-4328-
29/10
72/Ma/b12.74.5Rec4AdeM2NoNoAAPushInv5/0UlcII
50C54B-4407/1038/Fw/p632.19Asc6AdeM2NoNopreAPushInv9/1UlcIII
M=male,f=female,p/r/b=perectalbleeding,w/p=weakness,pallor,anorexia,mass=abdominalmass,a/b=alteredbowelhabit,Rec=Rectum,Asc=Asendingcolon,Tran=Transeverse
colon,Des=descendingcolon,sig=Sigmoidcolon,cae=caecum,Ade=Adenocarcinomausualtype,Muc=Mucinouscarcinoma.P=Present,A=absent,push=pushingmargin,Inf=
Infiltratingmargin,Ulc=Ulcerated,Exo=Exophytic,Inf=infiltrative.Inv=Involved,Til=tumourinfiltratinglymphocytes,Ext.Muc=Extracellularmucin,LVinvasion=Lymphovascular
invasion,PNinvasion=Perineuralinvasion.LN=lymphnode.
xvii
Thesis book
Thesis book
Thesis book
Thesis book
Thesis book

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Thesis book

  • 6. CLINICOPATHOLOGIC PATTERN AND DNA METHYLATION STATUS OF COLORECTAL CARCINOMA IN A GROUP OF BANGLADESHI PATIENTS Dr. Abu Khalid Muhammad Maruf Raza MBBS
  • 7. Contents Page _________________________________________________ no 1 INTRODUCTION 01 2 REVIEW OF LITERATURE a Anatomical consideration 04 b Physiology 08 c Tumors of the colon and rectum 09 3 MATERIALS AND METHODS 26 4 OBSERVATIONS AND RESULTS 33 5 DISCUSSION 54 6 SUMMARY AND CONCLUSION 62 7 BIBLIOGRAPHY 64 8 APPENDICES I Certificate from The Chicago University i II Clinical proforma ii III Preparation of stains and chemicals iv IV Tissue processing and staining procedures v V Protocol for DNA extraction viii VI Protocol for DNA methylation x VII TNM staging system xii VIII Master table xiv IX Illustrations xviii
  • 8. LIST OF TABLE Table no Title Page no 2.3:1 Genes silenced by hypermethylation in colorectal cancer 18 2.3:2 TNM Classification of Colorectal Carcinoma by American Joint Committee on Cancer (AJCC). 22 2.3:3 TNM staging for colorectal carcinoma. 23 4.1 Sex distribution in relation to age group. 35 4.2 Symptoms with tumour location of colorectal cancer cases. 36 4.3 Haemoglobin concentration in 50 cases of colorectal cancer patients 37 4.4 CEA (Carcinoembryonic antigen) level in 50 colorectal cancer cases 37 4.5 Site distribution of colorectal cancer from distal to proximal 38 4.6 Histological diagnosis of 50 cases of colorectal cancer 40 4.7 Histological grading of colorectal cancer cases with sex and location 41 4.8 TNM (Tumour, Node, Metastasis) stage with sex and location 42 4.9 Signet ring cells in colorectal cancer patients 45 4.10 Circumferential margin involvement, Perineural invasion and Tumour border configuration with TNM stage. 47 4.11 Selected genes that show methylation differences in 24 cases of Colorectal carcinoma. 53 5.1 Age and sex distribution of CRC in different studies and the present study. 54 5.2 Distribution of colorectal cancer in left colon and right colon. 57
  • 9. LIST OF FIGURES Fig. No Title Page no 2.1 Gross anatomy of large intestine 07 4.1 Bar diagram showing age distribution of colorectal cancer. 34 4.2 Growth pattern of colorectal cancer cases. 39 4.3 Various grade of tumour infiltrating lymphocytes in 50 cases. 43 4.4 Lymphovascular invasion in colorectal cancer patients 44 4.5 Extracellular mucin in colorectal cancer patient. 45 4.6 Scatter diagram showing total intensity in tumour tissue and its corresponding normal tissue in all 24 cases 48 4.7 Scatter diagram showing average beta of all 48 samples 49 4.8 Histogram showing tumour differential score. 50 4.9 Bar diagram showing sources of variation of methylation data. 51 4.10 Variation of methylation data due to tumour differentiation. 52
  • 10. 1.0 INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in the world and the second leading cause of cancer related deaths in the United States. Globally, the incidence of CRC varies widely with higher incidence rates in North America, Australia and Northern and Western Europe (Aljebreen, 2007). The lifetime risk of developing CRC is about 6% or one in 18. Over 95% of these CRC is adenocarcinoma (Kim et al, 2010). CRC is relatively uncommon in Indian sub continent. In India the incidence of colorectal cancer was found to be 4.2 and 3.2 per hundred thousand for male and female population respectively (Afroza et al, 2007). The incidence of colorectal cancer in Bangladesh is not exactly known, it appears to be common and occur in younger age group with slight male preponderence. Average age at diagnosis is 10 years less than the developed countries. Rectal bleeding is the most common symptoms and majority of the carcinoma were in the rectum (Hossain, 2007). The peak incidence of colorectal carcinoma is in between the age of 60 and 69 years. Fewer than 20% occur before the age of 50 years. Males are affected slightly more than females (Turner, 2010). Colorectal carcinoma are rare before the age of 40 without genetic predisposition or without predisposing factors (Hamilton, 2000). Early detection of colonic cancers is a challenging task as because clinical symptoms develop slowly. Per rectal bleeding is common. Many patients experience change in bowel habit (Yawe et al, 2007). Screening tests like digital rectal examination, simple laboratory 1
  • 11. investigations like estimation of CEA, estimation of haemoglobin, faecal occult blood test, and visualization of the gut mucosa by sigmoidoscopy and colonoscopy examination may be a help in the diagnosis (Aljebreen, 2007). Colorectal cancer is a multifactorial disease process. Etiology contributing from environmental factors including diatery factors, obesity, alcohol intake, smoking, life style and genetic and epigenetic abnormalities. The molecular events that leads to CRC is heterogenous and includes genetic and epigenetic abnormalities. Genetic events in colorectal cancer is genetic alteration of the APC gene, mutations in the KRAS and P53 gene and abnormalities in the DNA mismatched repair genes (Turner, 2010). Epigenetic changes, which is the heritable changes in gene function that are not due to changes in the DNA sequence is an important pathway in the mechanism of tumerogenesis in colorectal cancer. DNA methylation abnormalities is an important epigenetic changes in CRC and become an area of great interest in the field of cancer research. The significance of DNA methylation alteration in CRC has been reported widely. There is both hypermethylation and hypomethylation abnomalities in various genetic loci in CRC (Kim et al, 2010). Available data indicates some differences in CRC in Bangladeshi population like lower mean age of patients, predominently left sided tumour (Hossain, 2007). Correlation of histopathological features with clinical data and detection of genetic abnormalities can give better insight in this field. 2
  • 12. 1.1 AIM To see the morphologic pattern, clinical features and DNA methylation changes in colorectal carcinoma in a group of Bangladeshi patients. 1.2 OBJECTIVES 1. To see various histologic features of colorectal carcinoma. 2. To see different clinical presentation of colorectal carcinoma. 3. To see DNA methylation changes in colorectal cancer tissue and corresponding surrounding normal colonic tissue. 3
  • 13. 2.0 REVIEW OF LITERATURE 2.1 Anatomy of large intestine 2.1.1 Gross anatomy: The large intestine is about 1.5 meters long, extends from the distal end of ileum to anus. It begins in the right iliac fossa as the caecum, from which the vermiform appendix arises. The caecum becomes the ascending colon which passes upwards in the right lumbar region and hypochondrium to the inferior aspect of the liver. It then bends to the left forming the hepatic flexure (right colic flexure) and becomes the transverse colon. This loops across the abdomen with an anterior-inferior convexity until reaches the left hypochondrium. Where it curves inferiorly to form splenic flexure (left colic flexure) and becomes the descending colon. It then forms sigmoid colon in the left illac fossa. The sigmoid colon, descends deep into the pelvis and becomes the rectum, which ends in the anal canal at the level of pelvic floor (Borley, 2008). 2.1.2 Blood supply of the colon: Arterial supply: The arteries which supply the parts of the large intestine are from the colic branch of the superior mesenteric artery. The hind gut derivatives are supplied by the inferior mesenteric and rectal arteries (Borley, 2008). 4
  • 14. The venous drainage: The vein of the large gut are the corresponding superior and inferior mesenteric artery. The rectum and anal canal are drained by the rectal vein (Borley, 2008). Lymphatic drainage: The large gut is drained to the epicolic nodes, paracolic nodes, the intermediate colic and terminal nodes (Borley, 2008). 2.1.3 The nerve supply: The sympathetic supply of mid gut derivate are coeliac and mesenteric ganglia and the parasympathetic supply is by the vagus nerve. The hind gut derivatives are supplied by the superior hypogastric plexus. The rest of the portion is supplied by inferior hypogastrci plexus and pudendal nerve (Borley, 2008). 2.1.4 Developmental anatomy of colon: The caecum, the appendix, the ascending colon and the proximal two-third of the transverse colon are derived from the midgut. The distal one-third of the transverse colon, the descending colon, the sigmoid colon, the rectum and the upper portion of the anal canal are derived from hindgut (Sadler, 1995). 2.1.5 Histology of the colon: The large intestine is divided topographically into three segments ( Junquira et al, 1995): 5
  • 15. a. Colon, b. Rectum and c. Anal canal. All the segments histologically have four coats; a. Mucosa b. Submucosa c. Muscularis externa and d. Serosa (Young and Heath, 2000). The surface epithelium of the mucosa is made of tall columnar epithelium mixed with goblet cells turn downward as a tubular gland. The lower portions of the tubular glands have numerous goblet cells. The lamina propria is composed of fibrocollagenous tissue. The submucosa is composed of loose connective tissue containing large blood vessels and meissner plexus of nerve. Muscularis externa in the colon shows some variation from the other parts of the gut. The inner circular layer is complete and prominent. The outer longitudinal layer forms three equidistant, longitudinal bands (Tinia coli). Adventitia or serosa consists of thin connective tissue layer covered by the mesothelium (Yound and Heath, 2000). 6
  • 16. 7
  • 17. 2.2 Physiology of the large intestine The main function of the colon is absorption of water, Na+, and other minerals by removal of about 90% of the fluid. It converts the 1000-2000ml of isotonic chyme that enters it each day from the ileum to about 200-250ml of semisolid feces (Ganong, 2005). Motility and secretion of the colon: When peristaltic wave reaches to the valve, it opens and permits ilial chyme to squirt into the caecum. The movement of the colon include segmentation contraction and peristalsis. Segmentation contraction mixes the content of the colon and by exposing the more of the contents to the mucosa facilitates absorption. Peristaltic waves propel the contents towards the rectum (Ganong, 2005). Transit time in the colon: The first part of the test meal reaches the caecum in about 4 hours and all of the undigested food entered the colon in 8 to 9 hours. The first meal reaches the pelvic colon in 12 hours (Ganong, 2005). Absorption in the colon: Na+is actively transported out of the colon and water follows along the osmotic gradient. K+and HCO3 are secreted into the colon. The absorptive capacity of the colon makes it a suitable route of drug administration (Ganong, 2005). Daefecation: Distention of the rectum with faces initiates reflex contraction of the musculature and desire to defecate. The sympathetic nerve supply to internal anal sphincter is excitatory whereas the parasympathetic nerve supply is inhibitory (Ganong, 2005). 8
  • 18. 2.3 Tumours of the colon and rectum Many conditions, such as congenital disease, infection, inflammatory diseases, motility disorders and tumours affect the large intestine (Turner, 2010). The colorectal cancer is the second most common visceral cancer in the U.S.A. (Cooper, 1999). Virtually 98% of colorectal carcinoma are adenocarcinoma (Turner, 2010). The World Health Organization classified colorectal cancer histologically as follows (Hamilton. 2000). 2.3.1 WHO classification of tumors of colon and rectum: (Hamilton, 2000) 1. Non-neoplastic polyp i. Hyperplastic polyp ii. Peutz-Jeghers polyp iii. Juvenile polyp 2. Epithelial tumors 1. Adenoma i. Tubular adenoma ii. Villous adenoma iii. Tubulovillous adenoma iv. Serrated adenoma 9
  • 19. 2. Intraepithelial neoplasia (Dysplasia) associated with Chronic inflammatory diseases Low-grade glandular intraepithelial neoplasia High grade glandular intraepithelial neoplasia 3. Carcinoma i. Adenocarcinoma ii. Mucinous carcinoma iii. Signet ring carcinoma iv. Small cell carcinoma v. Adenosquamous carcinoma vi. Medullary carcinoma vii. Squamous carcinoma viii. Undifferentiated carcinoma 4. Carcinoid tumour 5. Mixed carcinoid-adenocarcinoma 6. Non-epithelial tumor i. Lipoma ii. Leiomyoma iii. Gastrointestinal stromal tumor 10
  • 20. iv. Leiomyosarcoma v. Angiosarcoma vi. Malignant lymphoma vii. Malignant melanoma 7. Secondary tumors 2.3.2 Colorectal carcinoma Colorectal cancer (CRC) is the third most common malignant neoplasm worldwide and the third most common malignancy and one of the leading causes of cancer death in women and men in the United States. The lifetime risk of CRC among women and men at average reaches 6% or one in 18 (Kim, 2010). The peak incidence of colorectal carcinoma is between 60 and 79 years. Fewer than 20% cases occur before the age of 50 years (Turner, 2010). 2.3.2.1 Incidence of colorectal carcinoma Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer related death in the United States (Aljebreen, 2007). In the USA and Western Europe, colorectal cancer constitutes approximately 10% of all malignancies (Leon et al, 2004). It represents 9.4% of all incident of malignancy in men and 10.1% in women globally. CRC is not equally common through out the world. The incidence varies in various geographical part of the world. High incidence seen in Europe, America, Australia, New Zealand with incidence ranges from 25.3/100000 to 45.8/100000 11
  • 21. population (Boyle and Langman, 2000). Compare to developed countries, lower incidence of colorectal cancer is seen in developing countries like Africa and Asia, including central and south Asia and India. Incidence ranges from 2/100000 to 8/100000 population (Notani, 2001). The epidemiological study of colorectal cancer done by Keating et al (2003) among 2272 pathological report in New Zealand showed the incidence of colorectal carcinoma was 46.8/1000000 for men and 43.5/100000 for women. Ayyub et al (2000), in a large hospital of Saudi Arabia has shown the incidence were 4.6/100000 in male and 4.4/100000 in female population. 2.3.2.2 Age and Sex distribution of Colorectal carcinoma Colorectal cancer is a disease of advanced aged population. Greater number of CRC are found in the six decades with a median age of about 62 years (Riddell, 2003). Keating et al (2003) done a study on the epidemiology of colon cancer in New Zealand. They found in their study that the mean age of women was 69.4 years and mean age of men was 68.5 years. Turner (2010) have shown nearly the same result in their study as the peak incidence of colorectal carcinoma was in between ages 60-70 and only 20% cases occur before the age of 50 years. Ayyub et al (2002) studied on clinicopathological trends in colorectal cancer on 160 cases in Saudi Arabia. They showed that the mean age was 56.3±14.98 years. Colorectal cancer is extremely rare in paediatric age group. Afroza et al (2007) reported a 11 year old Bangladeshi boy with primary mucinous adenocarcinoma in the rectum. 12
  • 22. Colorectal cancer affects men and women almost equally (Boyle and Langman 2000). Some study have shown male predominance (riddell et al, 2003). Keating et al (2003) showed in their study that the male and female ratio were almost equal. Fireman et al (2005) showed female predominance in their study as among 624 cases, 271 were male and 353 were female. 2.3.2.3 Clinical features of Colorectal carcinoma Patients with colorectal cancer have usually presented with abdominal pain, alteration of bowel habit, loss of weight, vomiting, frequently with colic, anorexia, bleeding per rectum, lump, indigestion and acute on chronic obstruction (Hamilton, 2000). Ayyub et al (2002) mentioned on their study that most of the patient presented with the symptom of abdominal pain, altered bowel habit, per rectal bleeding weight loss, intestinal obstruction and constipation. Duration of symptom varied from one month to 2 years. 2.3.2.4 Investigations in CRC The laboratory investigation include the blood and biochemical parameters like tumour markers (CEA, CA-19-9), and also the visualization of the lesion through endoscope. Ayyub et al (2002) studied clinicopathological trends in colorectal carcinoma showed anaemia (Hb% lOgm/dl or less) in 55% of the patients. The distribution of anaemia varied significantly in various sub site of primary colorectal carcinoma. About 30% patients had anaemia with left sided tumour and 70% of patients in right side colon cancer. They also measure CEA level in serum of colorectal cancer patients. CEA level varied from 1 to 13
  • 23. 850.2 with a mean of 48.62. Leon et al (2004) studied trend of incidence, subtype distribution and staging of colorectal carcinoma in the 15 years experience of a specialized cancer registry, showed more then 50% colorectal carcinoma can be detected by sigmoidoscopy and rest needed pancolonscopy for the choice of screening for individual at the risk of colorectal carcinoma. 2.3.2.5 Sub site distribution The right colon is considered from caecum to spleenic flexure; left colon includes descending colon, sigmoid colon and rectum (Gomez et al, 2004). The sub site-specific variation is seen in the development of colorectal carcinoma. Two-third of colorectal carcinoma occur in the left colon and the rest in the right colon (Leon et al, 2004). Ayyub et al (2002) showed majority of the colorectal carcinoma 68.2% in the rectum and sigmoid colon; and 22.5% colorectal carcinoma originating from ascending and transverse colon. Gomez et al (2004) showed total 31% of the cancer were in right side. The study also cited the differences in the anatomical distribution between sex, in female 48% were in right and 41% were left sided colon cancer in comparison to 59% cases with left colon cancer and 52% cases with right colon cancer in male. 2.3.2.6 Histological subtypes of colorectal carcinoma Most of the colon cancer is adenocarcinoma. In the study of Keating et al, (2003) showed 94.7% cases of adenocarcinoma, 4.2% were mucinous adenocarcinoma, 0.4% were signet ring cell carcinoma and less then 1% cases were adenosquamous, squamous and miscellaneous carcinoma. Hossain (2007) found 74% of cases were usual 14
  • 24. adenocarcinoma, 23% cases mucinous adenocarcinoma, 1.5% cases each signet ring cell carcinoma and small cell carcinoma. 2.3.3 Pathogenesis Environmental and genetic factors both contribute to colorectal cancer development. Genetic susceptibility may be the soil on which subsequent environmental factors act (Riddell, 2003). 2.3.3.1 Environmental factors: Epidemiologic studies have indicated that meat consumption, smoking, sedentary lifestyle and alcohol consumption are risk factors for colorectal carcinoma. Inverse associations include vegetable consumption, prolonged use of non-steroidal anti inflammatory drugs, oestrogen replacement therapy and physical activity (Hamilton, 2000). 2.3.3.2 Molecular carcinogenesis: The combination of molecular events that lead to colonic adenocarcinoma is heterogenous and includes genetic and epigenetic abnormalities (Turner, 2010). The genetic and epigenetic pathways involved are: 1. -catenin pathway which is associated with adenoma-carcinoma sequence. 2. Loss of p53. 3. K-ras mutation. 4. Delated in colon cancer gene (DCC gene). 15
  • 25. 5. Telomerase activity. 6. Microsatellite instability pathway which is associated with DNA mismatch repair. 7. DNA methylation abnormalities (Hypomethylation and Hypermethylation). 2.3.3.3 DNA methylation in cancer Cancer results from the accumulation of mutation in the genes. In addition to genetic mutation, this epigenetic change has been included as an alternative mechanism to cancer development. Epigenetics refers to change in the pattern of gene expression by mechanisms other than alterations in the primary nucleotide sequence of a gene(Herman, 2003). DNA methylation is the enzymatic attachment of methyl group to the 5th carbon of the cytosine base (Samarakoon, 2010). Methylation usually occurs in the CpG islands, a cytosine guanosine rich region in the DNA. In humans, DNA methylation is carried out by a group of enzymes called DNA methyltransferases. The letter “p” here signifies that the C and G are connected by a phosphodiester bond. Methylation patterns in tumor cells are significantly different from those in normal cells (Herman, 2003). Silencing or inactivation of tumour suppressor gene is seen in cancers mostly due to hypermethylation. Hypermethylation profiling over more than 15 tumor types (colon, stomach, pancreas, liver, kidney, lung, breast, ovary, endometrium, kidney, bladder, brain, and leukemia and lymphomas) has shown that all the metabolic pathways are affected by promoter hypermethylation-associated silencing. Hypomethylation leads to the inappropriate and increased levels of gene expression in tumors such as oncogene activation (Samarakoon, 2010). 16
  • 26. 2.3.3.4 DNA methylation in colorectal cancer: Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation (Kim, 2010). Hypermethylation in the CpG islands of the tumor suppressor gene promoters can lead to a complete block of transcription and inactivates the tumor suppressor genes. DNA hypomethylation could also drive neoplastic progression and transformation. It may make chromosomes more susceptible to breakage and oncogene activation (Kim, 2010). 2.3.3.4.1 Genes inactivated by promotor hypermethylation in colorectal cancer Hypermethylated genes are associated with colorectal neoplasia includes the tumour suppressor, mismatch-repair and cell-cycle regulatory genes (Wong, 2007). Table 2.3.1 shows functions and frequency of different genes involved by hypermethylation in colorectal cancer. 17
  • 27. Table: 2.3.1 Genes silenced by hypermethylation in colorectal cancer (Wong, 2007) Genes Function Frequency (%) APC Signal transduction, beta-catenin regulation 10–50 CDH13 Cell signalling (cell recognition and adhesion) 30–40 CDKN2A Cell-cycle regulation 15–30 CHFR Mitotic stress checkpoint 30–40 HIC1 Regulation of DNA damage responses 80 HPP1 Transmembrane transforming growth factor (TGF)- antagonist 80 LKB1 Cell signalling, cell polarity 5–10 MGMT Repair of DNA guanosine methyl adduct 30–40 MLH1 Genes Mismatch repair Function 10–20 p14ARF Cell-cycle regulation 20–30 RASSF1A DNA repair, cell-cycle regulation >50 SOCS1 Cell signaling 5–10 THBS1 Angiogenesis 10–20 TIMP3 Matrix remodelling, tissue invasion 10-30 18
  • 28. 2.3.3.4.2 DNA hypomethylation in colorectal cancer Hypomethylation may make chromosomes more susceptible to breakage and therefore lead directly to genomic instability. DNA hypomethylation can also lead to the activation of oncogenes such as S100A4 metastasis-associated gene in colorectal carcinoma and can lead to loss of imprinting (LOI) which can drive cellular proliferation in cancer. The clearest example of this phenomenon is hypomethylation of IGF2/H19 seen in about 40% of colorectal cancer tissue (Wong, 2007). Hypomethylation of CDH3 promotor, CD133, LINE-1 are also seen in colorectal cancer (Kim, 2010). 2.3.4 Gross morphology of colorectal carcinoma The tumor of the proximal colon tends to grow as polypoid and exophytic mass and in the distal colon the mass grow as annular and encircling manner. The gross morphology of the lesions are fungating. annular, tubular and ulcerated. The fungating growth is cauliflower like growth, some time ulceration is seen in the tip of the fungating growth. The annular growth encircling whole of the circumference and produce obstructive feature clinically. The tubular growth is flat (linitis plastica). The ulcerated growth form an ulcer in the gut wall. The superficial spreading lesion is difficult to identify grossly (Turner, 2010). The cut surface of the gut wall shows grayish white tissue replacing the bowel wall. Highly mucinous tumour have a gelatinous glaring appearance, and layers of mucus may separate the layers of the bowel wall (Turner, 2010). 19
  • 29. 2.3.5 Microscopic features of colorectal carcinoma 2.3.5.1 Adenocarcinoma: About eighty percent of colorectal carcinomas are histologically characterized by good gland formation and varying degrees of differentiation, from well to moderately differentiated. Well differentiated carcinoma have >95% glandular structure, moderately differentiated carcinoma have 50-95% glands, poorly differentiated carcinoma have 5-50% glandular structure and undifferentiated carcinoma have <5% glandular structures (Hamilton 2000) 2.3.5.2 Mucinous adenocarcinoma: Mucinous and Signet-ring adenocarcinoma account for 10% of colorectal cancer. This designation is used if >50% of the lesion is composed of mucin. This variant is characterized by pools of extracellular mucin that can contain malignant epithelium as acinar structure, strips of cells or single cells (Hamilton,2000) 2.3.5.3 Signet ring cell adenocarcinoma: Signet-ring and mucinous cell carcinoma account for approximately 10% of colorectal cancer. This variant of adenocarcinoma is definied by the presence of >50% of the tumour cells with prominant intracytoplasmic mucin (Hamilton, 2000) 2.3.5.4 Small cell carcinoma: A rare variant is small cell carcinoma which composes <1% of colorectal cancer (Cooper, 1999) 2.3.5.5 Adenosquamous carcinoma: These unusual tumours show features of both squamous carcinoma and adenocarcinoma either as separate areas within the tumour or admixed pattern (Hamilton, 2000). 20
  • 30. 2.3.5.6 Squmous cell carcinoma: Squamous cell carcinoma of the colon is very rare. To make diagnosis of the squamous cell carcinoma in the colon, there must be no other site of the squamous cell carcinoma in the body and no involvement of cloacogenic squamous lined mucosa (Rosai, 2004). 2.3.5.7 Medullary carcinoma: This rare variant is characterized by the sheets of malignant cells with vesicular nuclei, prominent nucleoli and abundant pink cytoplasm exhibiting prominent infiltration by intraepithelial lymphocytes (Hamilton, 2000). 2.3.5.8 Undifferentiated cancer: Undifferentiated cancer is uncommon, accounting for approximately 1 % of the colorectal carcinoma. They are malignant epithelial neoplasm that have no glandular structure or other features to indicate definite differentiation (Hamilton, 2000). 2.3.6 Staging of colorectal cancer: The major role of proper staging of colorectal carcinoma is to provide information to physician regarding patient's prognosis and the need for adjuvant therapy. For many years, pathologists used the classic Dukes' classification(1932), Astler-Coller classification(1954) and the TNM classification(Turner, 2010). Table 2.3.2 shows TNM classification and Table 2.3.3 shows TNM staging of colorectal carcinoma (Turner, 2010). 21
  • 31. Table:2.3.2 TNM Classification of Colorectal Carcinoma by American Joint Committee on Cancer (AJCC) (Turner, 2010). TUMOUR Tis In situ dysplasia or intramucosal carcinoma T1 Tumor invades submucosa T2 Tumor invades into, but not through, muscularis propria T3 Tumor invades through muscularis propria T3a Invasion <0.1 cm beyond muscularis propria T3b Invasion 0.1 to 0.5 cm beyond muscularis propria T3c Invasion >0.5 to 1.5 cm beyond muscularis propria T3d Invasion >1.5 cm beyond muscularis propria T4 Tumor invades adjacent organs or visceral peritoneum T4a Invasion into other organs or structures T4b Invasion into visceral peritoneum REGIONAL LYMPH NODES NX Lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in one to three regional lymph nodes N2 Metastasis in four or more regional lymph nodes DISTANT METASTASIS MX Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis or seeding of abdominal organs 22
  • 32. Table :2.3.3 TNM staging for colorectal carcinoma (Turner, 2010) Stage-I Tl N0 M0 or T2, N0, M0 Stage-II T3 N0 M0 or T4, N0, M0 Stage-III T any Nl M0 or T any,N2,N3, M0 Stage-IV T any N any Ml 2.3.7 Grading of colorectal cancer: Grading is done on the basis of differentiation of tumour cells. Differentiation refers to the extent to which neoplastic cells resemble comparable normal cells (Kumar et al, 2010). The formation of glands (acini) is the basis for grading system in CRC. Well differentiated carcinoma have >95% glandular structure, moderately differentiated carcinoma have 50- 95% glands, poorly differentiated carcinoma have 5-50% glandular structure and undifferentiated carcinoma have <5% glandular structures. Mucinous carcinoma and signet ring cell carcinoma by definition are poorly differentiated cancer (Hamilton, 2000). 2.3.8 Prognosis of colorectal cancer: (Rosai, 2004) There are several clinical and pathological parameters which determines prognosis. Most important is the tumour stage. 5 years survival rate in TNM stage I disease is 90 to 98 percent. About 80 percent 5 year survival in TNM stage II and less than 10 percent in stage III with many lymph nodes involvolvement and in stage IV (Riddell, 2003). Some factors related to prognosis are given below: 23
  • 33. Age: Tumour occuring in very young and old patient have poor prognosis. Sex: The prognosis is better in female than in male. CEA serum level: >5.0 ng/ml have been shown to have adverse prognosis. Tumour location: Tumour located in the sigmoid colon and rectum have the worse prognosis. Local extent: Lymph node metastasis have worse prognosis than local invasive. Tumour size: There is a little relationship between the size of the tumour and prognosis. Tumour edge: The non-polypoid edge of the tumour have the worse prognosis than polypoid cancer. Obstruction: Obstruction is an indicator of worse prognosis. Perforation: It is due to invasion to the wall shows worse prognosis. Tumour margin and inflammatory reaction: Pushing margin with inflammatory response have better prognosis. Vascular invasion: When venous invasion is present, the 5-years survival decreases markedly. Perineural invasion: Reflects the advanced stage of the disease. Surgical margins: Presence of tumour in surgical margin is the single most critical factor for recurrence of carcinoma. 24
  • 34. Tumour thickness: Central depressed area of the tumour is correlated with the presence of lymph node and liver metastasis and also with bad prognosis. Microscopic tumour type: Mucinous carcinoma, Signet ring cell carcinoma and anaplastic carcinoma have worse prognosis. Acinar morphology: Microacinar pattern of growth is associated with poor prognosis. Tumour angiogenesis: Tumour angiogenesis predict recurrence and is associated with decreased survival in colorectal carcinoma. Mucin-related antigen: Colorectal carcinoma that express the mucin-associated antigens sailyl-Tn and sailyl-Lewis antigen have more aggressive clinical course. HLA-DR expression: Patient with tumour having strong HLA-DR expression show better prognosis. HCG expression: Effect as adverse indicator of prognosis. Bcl-2 protein expression: Associated with better prognosis. DNA ploidy: The DNA aneuploidy and risk of recurrence of survival. Allelic loss of chromosome 18q: Have strongly negative prognostic significance. 25
  • 35. 3. MATERIAL AND METHOD 3.1 Place and period of study a. The clinical and histopathological part of this study was carried out at the Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka during the period of November 2009 to July 2010. A total of 50 cases of colorectal adenocarcinoma from all ages and both sexes were included in the study. b. The DNA methylation part of this study performed on 48 samples (24 tumour tissue and 24 corresponding healthy tissue) was carried out in collaboration at the Department of Health Studies, Biological Sciences Division (BSD) of The University of Chicago Medical Center, Chicago , IL , USA (Appendix-I). 3.2 Collection of the case A total of 50 cases of colorectal carcinoma were collected for study. For selection, inclusion criteria described below was followed. Cases were collected from Bangabandhu Sheikh Mujib Medical University (BSMMU) and private hospitals and clinics in Dhaka city. 3.3 Inclusion criteria a. Histologically confirmed cases of adenocarcinoma of the colon. b. Cases with complete clinical information. c. Availability of fresh unfixed colorectal cancer specimen. d. Availability of normal uninvolved colonic tissue away from the tumour. 26
  • 36. 3.4 Exclusion criteria a. Clinically suspected colorectal carcinoma subsequently proved to be non-malignant lesions after histological examination. b. Non Hodgkin lymphoma and other non epithelial tumors of the colon. c. Cases without clinical data. d. Formalin fixation or delay in receiving specimen. 3.5 Collection of clinical information Detailed clinical information was obtained by taking history and recorded in clinical proforma (Appendix-II). Filling up of the clinical proforma was performed in all cases either from patient's attendants statement and/or from patients file. 3.6 Collection of the tissue block for histopathological examination Fresh unfixed specimen were obtained after surgical resection. Large plastic container with lid were used for collection. Containers were properly labeled with identification number, name of the patient, address, type of specimen and date and place of operation. 3.6.1 Fixation of sample for Histopathological examination: After collecting blocks for methylation study (describeb next), the unfixed specimen were transferred to 10% formalin for overnight fixation. The next morning the specimen were examined during gross cut up and tissue blocks were taken according to the following procedure (Rosai, 2004) 27
  • 37. a. Recording of gross features of the specimen: i. Specimen type, length of the specimen and amount of mesentery. ii. Tumour characteristics: Tumor size including thickness, extent around bowel, tumour configaration(exophytic, infiltrative, ulcerating), presence of necrosis or hemorrhage, tumour number, extent through bowel wall, serosal involvement, invasion of adjacent organs and distance of the tumour from the proximal and distal resection margins. iii. Number of lymph nodes found and size of the largest node. b. Sections of tissue blocks: i. Three blocks from tumour including the serous layer. ii. One block from tumour with surrounding normal mucosa. iii. Proximal line of resection. iv. Distal line of resection v. Appendix, if included in the specimen. vi. All the lymph nodes. vii. Polyp/ Suspicious areas. 3.6.2 Tissue processing for routine histopathological examination: All the tissue blocks were submitted for routine processing and paraffin Embedding (appendix-III). The tissue processing and staining was performed following the standard protocol (appendix-IV). Microscopic examination of routine paraffin sections stained with haematoxyline and eosin staining method were carried out during which relevent points were recorded. 28
  • 38. 3.6.3 Routine microscopic examination Routinely stained sections examined first under low power and then under high power magnification. The following points were noted during examination: i. Histologic type of growth. - Adenocarcinoma(NOS). - Mucinous. - Signet ring cell. - Small cell. ii. Histologic grade (applicable in adenocarcinoma (NOS) type). - Well differentiated. - Moderately differentiated. - Poorly differentiated. iii. Extent of invasion. -Submucosa. -Muscle coat. -Serosa. -Pericolic fat. iv. Proximal and distal resection margins. v. Circumferential ( radial) margin. vi. Lymphatic (small vessel) invasion. vii. Venous (large vessel)invasion. viii. Perineural invasion. ix. Tumor border configaration - Pushing. 29
  • 39. - Infiltrative. x. Lymphocytic response Grade 0 - <10 lymphocytes per high-power field (HPF). Grade 1- Tumour infiltrating lymphocytes (TIL) level of 10–50 HPF. Grade 2 -Tumour infiltrating lymphocytes (TIL) level 50-100 per HPF. Grade 3- Tumour infiltrating lymphocytes (TIL) level >100 per HPF. xi. Extracellular mucin content -No extracellular mucin. -1-49% extracellular (Tumour cell) mucin. ->50% extracellular (Tumour cell) mucin. xii. Signet ring cells (Ogino et al, 2008) - No signet ring cell. - 1-49% of tumour cell. - >50% of tumour cells. xiii. Lymph node status- number of lymph nodes involved. The tumour was classified according to the World Health Organization classification (Hamilton, 2000). The tumour staging was done using TNM classification (Turner, 2010). 30
  • 40. 3.7 DNA methylation study 3.7.1 Tissue sample collection for DNA methylation study Paired unfixed tissue samples, one each from tumour and healthy mucosa were taken in DNAse free 1.5 ml eppendorf tube (Ambion Catalogue # AM12450, Ambion company, USA). Dimention of the tissue blocks were 4 to 5 mm. These eppendorf were kept in -20°c and sent to the department of Health Studies, Biological Sciences Division of The University of Chicago Medical Center, Chicago , IL , USA in dry ice. DNA extraction and methylation work done at the Department of health studies (BSD), The University of Chicago, Chicago, IL, USA under aggrement between the department of pathology, BSMMU and Department of health studies (BSD), The University of Chicago, Chicago, IL, USA. 3.7.2 DNA extraction from colonic tissue: DNA extraction were done using Gentra Purgene Tissue kit (Qiagen Catalogue # 158622, Qiagen company, USA) (Appendix-V). 3.7.3 DNA Methylation by Infinium Assay: Principle: The DNA sample is first treated with sodium bisulphite which converts the unmethylated cytosine bases to uracil. The methylated cytosines on the other hand, remains unaffected. DNA methylation in this study was based on Infinium assay 31
  • 41. system using The Human Methylation 27 BeadChip (Inf HumanMeth27, RevB BC Kit, Catalogue # WG-311-1201 introduced by Illumina Inc, USA) ( Appendix-VI). 3.8 Statistical analysis: Histopathological portion: All the necessary and relevant data were recorded methodically and meticulously as far as possible in the clinical proforma. Relevant data were analyzed by standard statistical method. DNA methylation portion: The raw image data was processed by Bead Studio software provided by Illumina inc. USA, to generate the average beta values. Average beta is the methylation status of a gene locus. It is calculated by intensity of the methylated sequence(x)/ [intensity of the methylated sequence (x)+ intensity of the unethylated sequence (y) ]. The ratio is between 0 to 1, where 0= extremely hypomethylated and 1= extremely hypermethylated. Unpaired t-test was done to compare the average beta between the tumour & normal tissue. DiffScore was calculated from the p-value of the un-paired t-test. Delta Beta was calculated as beta of tumor tissue minus beta of normal tissue. 32
  • 42. 4.0 OBSERVATIONS AND RESULTS This cross sectional study was undertaken to see the morphologic pattern and DNA methylation changes in colorectal carcinoma in a group of Bangladeshi patients. Clinical data were recorded. DNA methylation of non tumorous areas of colon of the same patient was also determined. A total of 50 cases of colorectal cancer with their corresponding normal mucosa were included in the study. As molecular genetic laboratory facility is not available at present in BSMMU, the DNA methylation part was done in the molecular biology laboratory of the Department of Health Studies (BSD) of the University of Chicago,Chicago, IL, USA with aggrement between two department (Appendix-I). 4.1 Age and sex distribution 4.1.1: Age distribution of the colorectal carcinoma cases The age range was from 19 years to 84 years with a mean age of 46.6 ± 14.8 years. The patients were divided into 8 groups on the basis of decades. Out of 50 cases maximum number 12 (24%) of patients belonged to the age group 50-59 years, followed by 11(22%) cases each in 30-39 years and 40-49 years groups, 6(12%) cases in 20-29 years group, 5 (10%) cases in 60-69 years group, 3 (6%) in 70-79 years group and 1 (2%) case each in 10-19 years and 80-89 year groups. Figure 4.1 shows age distribution of colonic cancer patient. From this figure, peak age of CRC appears to be 30 to 60 years in our population.
  • 43. Figure 4.1 : Bar diagram showing age distribution of colorectal cancer. 4.1.2 Male to female ratio In this study out of total 50 cases, 29 (58%) cases were male and 21 (42%) cases were female with male to female ratio of 1.4:1, male patients were higher than female patients. Table 4.1 shows male and female patients with CRC in different age groups.
  • 44. Table 4.1 : Sex distribution in relation to age group. Age in years Total number of cases No. of Male No. of Female 10-19 1(2%) 1(2%) - 20-29 6(12%) 4(8%) 2(4%) 30-39 11(22%) 5(10%) 6(12%) 40-49 11(22%) 6(12%) 5(10%) 50-59 12(24%) 7(14%) 5(10%) 60-69 5(12%) 3(6%) 2(4%) 70-79 3(6%) 3(6%) - 80-89 1(2%) - 1(2%) Total=8 50(100%) 29(58%) 21(42%) 4.2 Clinical presentation at the time of attending to doctor At the time of first consultation, majority of the patients 20(40%) had per-rectal bleeding, 8(16%) had abdominal pain, 7(14%) had altered bowel habit, 6(12%) had generalised weakness, anorexia and pallor, 6(12%) had combined per rectal bleeding and abdominal pain and 3(8%) had palpable abdominal mass. The duration of symptoms ranged from one month to 24 months. Table 4.2 shows clinical presentation of colorectal cancer cases.
  • 45. Table 4.2: Symptoms with tumour location of colorectal cancer cases. Symptoms Cases N=50 Cases in different location Right colon Left colon Per rectal bleeding 20 (40%) 1 19 Abdominal pain 8 (16%) 6 2 Altered bowel habit 7 (14%) 4 3 Generalized weakness, anorexia and pallor 6(12%) 4 2 Per rectal bleeding and abdominal pain 6(12%) 4 2 Palpable abdominal mass 3(6%) 2 1 4.3 Haemoglobin concentration in blood Haemoglobin levels were recorded in 50 cases. It ranged from 8.3 to 14.6 gm/dl. Haemoglobin level <10gm/dl considered anaemia in both sex (Ayyub,2002). Haemoglobin was below 10 gm/dl in 13 (26%) cases and in 37(74%) cases it was more than 10 gm/dl. In 13 cases of below 10gm/dl, 8(62%) were female and 5(38%) were male. In 37 cases of hemoglobin level more than 10gm/dl, 24(65%) cases were male patient and 13(35%) were female. Table 4.3 shows haemoglobin concentration in blood of 50 colorectal cancer patient.
  • 46. Table 4.3: Haemoglobin concentration in 50 cases of colorectal cancer patients Hb (gm/dl) N=50 Male Female <10 13(26%) 5(38%) 8(62%) >10 37(74%) 24(65%) 13(35%) 4.4 CEA (Carcinoembryonic antigen) level in 50 colorectal cancer cases CEA levels were recorded in 50 cases. It ranged from 0.31 to 672 ng/ml. CEA <5ng/ml considered normal (Aljebreen, 2007). In 25 (50%) cases CEA was below 5 ng/ml and in 25 (50%) it was more than 5 ng/ml (Table 4.3). Table 4.4: CEA (Carcinoembryonic antigen) level in 50 colorectal cancer cases CEA (ng/ml) N=50 Male Female <5 25 15(30%) 10(20%) >5 25 19(38%) 6(12%)
  • 47. 4.5 : Sub site distribution of colonic cancer Distribution of cancer among 50 cases according to the site of the colon affected were as follows: 33 (66%) case were in rectum, 6 (12%) were in the ascending colon , 3 (6%) in hepatic flexure of colon, 2 (4%) each in caecum, descending colon, sigmoid colon and in the transverse colon (Table 4.5). Of the total 50 cases 37 (74%) were in the left colon (splenic flexure to rectum) and 13 (26%) were in the right colon (caecum to upto splenic flexure). Distribution of colon cancer in 29 male cases were : 17 (34%) in rectum, 4 (8%) in ascending colon, 2 (4%) each in transverse colon, hepatic flexure of colon and caecum and one(2%) each in sigmoid colon and descending colon. In 21 female cases, 16 (32%)cases were in rectum, 2 (4%) cases in ascending colon, one(2%) case each in sigmoid colon, descending colon and hepatic flexure of colon. Table 4.5: Site distribution of colorectal cancer from distal to proximal Variable N=50 Male Female Rectum 33 17(34%) 16(32%) Sigmoid colon 2 1(2%) 1(2%) Descending colon 2 1(2%) 1(2%) Transverse colon 2 2(4%) - Hepatic flexure of colon 3 2(4%) 1(2%) Ascending colon 6 4(8%) 2(4%) Caecum 2 2(4%) -
  • 48. 4.6 Size and shape of the lesion Morphology of the growth were recorded by examining the surgically resected specimen. Maximum diameter of the growth ranged form 3 cm to 10 cm. Grossly 25 (50%) tumours were ulcerated growth, 22 (44%) were polypoid or exophytic growth and 3(6%) were infiltrative or annular growth. Figure 4.2 shows growth pattern of colorectal cancer patients. Figure 4.2 Growth pattern of colorectal cancer cases. 4.7 Histological features of colorectal cancer cases In 50 cases of colorectal adenocarcinoma, 44(88%) were adenocarcinoma (NOS) and 6(12%) were mucinous adenocarcinoma. Of 44 cases of adenocarcinoma (NOS), 23 (46%) were male and 21 (42%) cases were female. All 6 (12%) cases of mucinous adenocarcinoma were male (Table 4.6). Ulcerating Exophytic Infiltrative
  • 49. Table 4.6: Histological diagnosis of 50 cases of colorectal cancer Histological type N=50 Male Female Adenocarcinoma 44 (88%) 23 21 Mucinous adenocarcinoma 6(12%) 6 - 4.7.1: Histological grading of colorectal cancer Of 50 colorectal cancer cases, 39(78%) cases were moderately differentiated and 11(22%) cases were poorly differentiated. In this present study no well differentiated adeno carcinoma was observed. All the mucinous carcinoma were included in the poorly differentiated group (Hamilton, 2000). Male predominance was observed in both moderately differentiated and poorly differentiated cases. Among the 39 moderately differentiated cases, 22 cases were seen in male and 17 cases in female and of 11 poorly differentiated cases, 7 cases were seen in male and 4 in female. Of thirty nine moderately differentiated cases, 10(20%) cases originated in right colon and 29 (58%) cases in the left colon. Of 11 poorly differentiated carcinomas, 4(8%) cases were in the right colon and 7(14%) cases were in the left colon. Table 4.7 shows histological grade of colorectal cancer in 50 cases with location and sex distribution.
  • 50. Table 4.7: Histological grading of colorectal cancer cases with sex and location: Tumour differentiation Number Male Female Right colon Left colon Moderately differentiated 39(78%) 22(44%) 17(34%) 10(20%) 29(58%) Poorly differentiated 11(22%) 7(14%) 4(8%) 4(8%) 7(14%) 4.7.2 Clinical stage of cases of colorectal cancer Staging of colonic cancer were done in all the cases. It was done on the basis of TNM system (Turner, 2010). The maximum number of cases 22 (44%) were in stage III, 14 (28%) cases were in stage II and 13 (26%) cases in stage I and 1 (2%) case in stage IV. Out of 22 stage III cases, 11(22%) cases were male and 11(22%) cases were female. In 14(28%) cases of stage II, 9(18%) cases were male and 5(10%) cases were female. In stage I there was a total 13(26%) cases, 8(16%) cases were male and 5(10%) cases were female. One (2%) stage IV case were male patient. Table 4.8 shows TNM stage in 50 colorectal cancer cases with sex distribution and location.
  • 51. Table 4.8: TNM (Tumour, Node, Metastasis) stage with sex and location TNM stage Number of cases Male Female Right colon Left colon Stage I 13(26%) 8(16%) 5(10%) 2(4%) 11(22%) Stage II 14(28% 9(18%) 5(10%) 6(12%) 8(16%) Stage III 22(44%) 11(22%) 11(22%) 8(16%) 14(28%) Stage IV 1(2%) 1(2%) - 1(2%) - TNM=Tumour Node Metastasis 4.7.3 Lymph node metastasis in 48 cases of colorectal cancer Out of 50 resected specimen, 49 cases contained lymph nodes. Lymph nodes number ranged from four to twenty five. Histologically lymph node metastasis were present in 23(47%) cases. Among the 23 nodal metastasis cases, 17(74%) cases metastasized from moderately differentiated primary tumour and 6(26%) cases from poorly differentiated primary carcinoma.
  • 52. Nil Grade-1 Grade-2 Grade-3 4.7.4 : Tumour infiltrating lymphocytes (TIL) in colorectal cancer patients Tumour infiltrating lymphocytes (TIL) were observed in all the 50 colorectal cancer cases and catogorized into four groups, nil or absent, grade- 1, grade-2 and grade- 3. 20(40%) cases were in grade -1, 18(36%) cases were in grade- 2, 4(8%) cases were in grade- 3 and the rest 8(16%) cases has no tumour infiltrating lymphocytes. Figure 4.3 show percentage of tumour infiltrating lymphocytes in 50 colorectal cancer cases. Figure 4.3: Various grade of tumour infiltrating lymphocytes in 50 cases.
  • 53. Present Absent 4.7.5: Lymphovascular invasion in colorectal cancer patients Lymphovascular invasion was present in 16(32%) cases and absent in 34 (68%) cases Figure 4.4 lymphovascular invasion in CRC cases. Figure 4.4: Lymphovascular invasion in 50 colorectal cancer cases 4.7.6: Extracellular mucin in colorectal cancer patients Out of 50 colorectal cancer patients percentage of extracellular mucin in the tumour tissue were recorded. 1-49% areas of extracellular mucin was present in 18(36%) cases, >50% extracellular mucin was present in 6(12%) cases and no extracellular mucin was found in 26 (52%) cases. Figure 4.5 shows distribution of extracellular mucin in colorectal cancer cases.
  • 54. Figure 4.5 Extracellular mucin in colorectal cancer patient. 4.7.8: Signet ring cells in 50 colorectal cancer cases Percentage of signet ring cells in the tumour tissue were recorded. 1-49% of the tumour cells were signet ring cells in 17(34%) cases and no signet ring cells in 33 (66%) cases. No cases having more than 50% signet ring cells was present in this series. Table-4.9 shows percentage of signet ring cells in 50 colorectal cancer cases Table 4.9: Signet ring cells in colorectal cancer patients Percentage of signet ring cells Number of cases (%) Nill (%) 33(66%) 1-49% 17(34%) >50% -
  • 55. 4.7.9: Circumferential margin, perinural invasion and tumour border configuration in colorectal cancer cases Histologically out of 50 colorectal cancer cases circumferential margin were involved in 12 (24%) cases. Of twelve involved cases 8 (67%) were stage in III and 4 (33%) were in stage II disease. In uninvolved 38 cases, 14 (37%) cases were in stage III, 13 (34%) cases were in stage I, 10 (26%) were in stage II and one (3%) case in stage IV. Out of 50 colorectal cancer cases, perineural invasion was present in 15(30%) cases. Out of these 15 cases 10 (66%) cases had stage III disease, 3 (20%) cases had stage II disease, one (7%) case each in stage I and stage IV. Perinural invasion was absent in 35(70%) cases. In which 12(24%) cases each were in stage I and stage III and 11(22%) cases in stage I disease. In 50 cases, tumour border configurations were examined microscopically. Infiltrative pattern were seen in 26 (52%) cases and pushing pattern in 24(48%) cases. In 26(52%) infiltrative pattern cases, 15 (58%) cases were in stage III, 6 (23%) cases in stage II, 4 (15%) cases in stage I and one (4%) case in stage IV. In 24 cases having pushing border, 9 (37%) cases were in stage I, 8 (33%) cases in stage II, 7 (33%) cases in stage IV. Table 4.10 shows Circumferential margin involvement, Perineural invasion and Tumour border configuration with TNM stage
  • 56. Table 4.10: Circumferential margin involvement, Perineural invasion and Tumour border configuration with TNM stage. TNM stage Circumferential margin Perinural invasion Tumour border configuration Involved N=12 Not involved N=38 Present N=15 Absent N=35 Infiltrative N=26 Pushing N=24 Stage-1 - 13(34%) 1(7%) 12(34%) 4(15%) 9(37%) Stage-2 - 10(26%) 3(20%) 11(32%) 6(23%) 8(33%) Stage-3 4(33%) 14(37%) 10(66%) 12(34%) 15(58%) 7(30%) Stage-4 8(67%) 1(3%) 1(7%) - 1(4%) - 4.8 DNA Methylation in 24 colorectal cancer tissue and corresponding normal colonic tissue Genome-wide methylation status was interrogated at nucleotide resulotion in 27500 CpG loci. In figure 4.6 scatter diagram shows a linear correlation between the total intensity (methylated + unmethylated) of tumour and normal tissue. Strong positive correlation was seen which indicates there is no bias in the microarray intensity data. Figure 4.6 shows total intensity of ethylation data in all 24 cases.
  • 57. Figure 4.6: Scatter diagram showing total intensity in tumour tissue and its corresponding normal tissue in all 24 cases 4.8.1 Methylation changes in tumour and surrounding normal tissue There was significant difference in the methylation status between tumour and sorrounding normal tissue. 4.8.1.1 The average beta Average beta is the methylation status of a gene locus. It is calculated by intensity of the methylated sequence(x)/ [intensity of the methylated sequence (x)+ intensity of the unethylated sequence (y) ]. The ratio is between 0 to 1, where 0= extremely hypomethylated and 1= extremely hypermethylated. Figure 4.2 shows scatter plot of average beta for tumour tissue against that of corresponding normal tissue in 24 cases. The figure shows that in the tumor tissue, there are a number of loci that are hypomethylated compared to the corresponding normal tissue and a number of loci that are
  • 58. hypermethylated. The central line represents regression line and other two lines on either side represents the boundary for two fold change Fig 4.7: Scatter diagram showing average beta of all 48 samples 4.8.1.2 Tumour differential score Tumour differential score indicates how significant the differenceis between the methylation status of the normal and tumour tissue. The more the positive value, the more that is hypermethylation. The more negative the more hypomethylation. Usually more than 30 is significant and indicates hypermethylation. Figure 4.4 shows some cases beyond +30 which are hypermethylated loci and many data beyond -30 which indicates hypomethylated loci. It also shows majority of the data clustering around 0 which indicates no differential methylation pattern in many of the cases.
  • 59. Figure 4.8: Histogram showing tumour differential score. 4.8.2 Factors responsible for the change in methylation data Bar diagram ( Fig 4.6) shows the significance of the different sources of variation in the total methylation data done by ANOVA test. It indicates the factors that influence the variation of the methylation data in 48 samples. It shows significance of factors responsible for the methylation changes in tumour tissue and normal tissue . Difference of the tissue (whether tumour or normal), sex (male or female, case id i.e case to case variation and location of the lesion (proximal or distal colon).
  • 60. Fig 4.9: Bar diagram showing sources of variation of methylation data. Another important factor that is responsible for the changes in the methylation is tumour differentiation. In this study in 24 cases 6 cases were poorly differentiated tumour and rest were moderately differentiated tumour. There were changes in methylation pattern due to differentiation (Figure 4. 7).
  • 61. Figure 4.10 Variation of methylation data due to tumour differentiation. 4.8.3 Genes involved in the methylation difference in colonic tumour tissue and normal colonic tissue Some of the candidate genes are identified in this study which show methylation difference in case of tumour tissue compared to normal colonic tissue. But which of these hypo or hypermethylated genes play role in CRC and what are their exact role are yet to be determined and needs further study. Table 4.11 show some genes that are methylated in colorectal cancer in this study.
  • 62. Table 4.11: Selected genes that show methylation differences in 24 cases of Colorectal carcinoma. Genes Tumour AVG. Beta Normal AVG Beta DiffScore Delta Beta EYA4 0.516264 0.149085 362.88 0.367179 TFPI2 0.422315 0.100107 362.88 0.322208 GATA4 0.569414 0.243687 362.88 0.325726 CDKN2A 0.422516 0.159246 362.88 0.263270 DCC 0.566206 0.289243 362.88 0.276963 MDF1 0.550374 0.083746 362.88 0.461493 ITGA4 0.630950 0.122244 362.88 0.508707 GAD2 0.430238 0.155448 362.88 0.274790 ADCY4 0.474352 0.151537 362.88 0.322815 CCNA1 0.663816 0.300486 362.88 0.363330 DAB21P 0.455048 0.036997 362.88 0.41805
  • 63. 5.0 DISCUSSION In this study, 50 cases of colorectal cancer were analyzed to find out histomorphological features, selected clinical data and DNA methylation changes in 24 cases. The mean age of the 50 cases was 47±14.8 years. The age range was from 19-84 years with male and female ratio of 1.4:1. 58% of the cases were below the age of 50 years. Peak incidence of colorectal cancer in this study were 50-59 years which is lower than that of western and other countries (Table 5.1). Turner (2010) found only 20% of the cases below 50 years. Keating et al (2003) found only 6.3% cases below 50 years. (Table-5.1). Table 5.1 Age and sex distribution of CRC in different studies and the present study. Investigator/ Country Peak incidence (Years) Mean age (Years) Range of age (Years) Cases <50 years Male : female Turner (2010),USA 60-70 <20% 1.2:1 Gomez et al (2004), UK 60-70 70±11 01-90 1.5 1 Riddel et al (2003), USA 60-70 60 3 2 Keating et al (2003), New Zealand 70-80 69 6.3 Equal Hossain(2007) Bangladesh 40-49 44.1±16.2 11-75 53% 1.6:1 Present study 50-59 46.6±14.8 19-84 58% 1.4:1 54
  • 64. The mean age of colorectal cancer in this present study indicate that colorectal carcinoma is relatively common in lower age group in our country. Though incidence of colorectal cancer in Bangladesh is not exactly known, it appears to be common in younger age group. This may be due to both environmental factors and genetic factors. Leon et al (2004) commented on recent trends in colorectal cancer. They observed rapid increase of CRC in the developing countries. Gomez et al (2004) commented a tendency of right sided shift from left sided colorectal cancer distribution. Keating et al (2003) suggested right sided cancer to be less well differentiated than the left sided CRC. Presently we do not know whether the CRC changing trends in Bangladeshi population i.e early age of onset, Right colon shift etc. It was also observed in present study that there is slight male preponderance regarding colorectal cancer cases. Keating et al (2003) found equal gender distribution in their study. Rectal bleeding was the commonest presentation (40% cases) at the time of first consultation. The other presenting complaints were abdominal pain (16% cases), altered bowel habit (12% cases), anorexia, pallor and generalized weakness (12% cases). Palpable abdominal mass, partial intestinal obstruction and weight loss were present in addition to the major complaints. Though abdominal pain has been described by some to be the commonest symptom in colorectal cancer (Aljebreen, 2007), it was not the case in this present study. Only 8(16%) cases had this feature. One possible explanation is negligence on the part of the patient and frequent use of analgesics. In this study, 20(40%) patients presented with per rectal bleeding, is similar to other study (Hossain, 2007). Per rectal bleeding was observed in 17(85%) cases of left colon cancer. 55
  • 65. Most of the cases with per rectal bleeding, tumour was present in rectum which explains partly that per rectal bleeding may be most common symptoms in rectal cancer. Anaemia was present in 13(26%) cases. Among these anaemic patients with colorectal cancer 8 cases were female and 5 cases were male. About 69% of cases with cancers in the right sided colon were anaemic and 31% of the patients with left sided cancer had anaemia. Ayyub et al(2002) observed anaemia in 55% of their cases and also mentioned 70% cases were in right sided colon and 30% cases in left sided colon. This present study shows lower haemoglobin levels for right sided colon cancer. CEA (Carcinoembryonic antigen) level in serum were ranged from 0.39 ng/ml to 672 ng/ml with mean level 26.6. CEA was elevated (>5 ng/ml) in 25(50%) cases. Rosai (2004) commented CEA more than 5 ng/ml is the adverse prognosis factor in CRC. In this present study, in 25 cases having CEA more than 5 ng/ml, 17(68%) cases were in stage III and one case was in stage I. This shows majority of the patients having elevated CEA are in high stage disease. Aljebreen (2007) found 32% of cases of colorectal cancer with elevated CEA level. Ayyub et all (2002) found CEA level ranged from 1 to 850.2 ng/ml with a mean of 48.62. The sub site distribution of colorectal cancer in this study shows 37(74%) cases were in the left colon and 13(26%) cases in the right colon. Recent trend of shifting of CRC towards right colon observed by Gomez et al (2004) is not supported by this present study. However it is similar to the studies done by other researchers. This is shown in table 5.2. 56
  • 66. Table-5.2: Distribution of colorectal cancer in left colon and right colon. Investigator Left colon Right colon Riddell et al(2003), USA 75% 25% Leon et al (2004), Italy 70% 30% Ayyub et al (2002), Saudi Arabia 70% 30% Hossain(2007), Bangladesh 63% 37% Present study 74% 26% In Among 37 cases of left colon cancer cases, 33 cases were in rectum. This shows higher percentage of rectal cancer in this study. Size and shape of the tumour were recorded in all 50 cases. The size of the tumour ranged from 3 cm to 10 cm. Pattern of growth were, 25(50%) cases ulcerating, 22(44%) cases exophytic/polypoid and the rest 3(6%) infiltrative. 18(72%) of the ulcerated lesion was present in rectum. This may be one of the probable cause of per rectal bleeding in most of the rectal cancer cases. On histological examination of 50 colorectal cancer cases, 44(88%) of cases were usual adenocarcinoma and 6(12%) cases were mucin secreting adenocarcinoma. Among these 39(78%) cases were moderately differentiated and 11(22%) cases were poorly differentiated cancer. No well differentiated cancer was reported in this series. Ekem et al (2008) found 37% moderately differentiated, 17% well differentiated and 6% poorly differentiated cancer. Keating et al (2003) found right sided tumour were less well differentiated than left sided tumour. In this study. of thirty nine moderately differentiated cases, 10(26%) originated in right colon and 29 (74%) in the left colon. Of the 11 poorly 57
  • 67. differentiated carcinomas, 4 were in the right colon and 7 were in the left colon. In this study left colon cancer were less well differentiated than right side. Tumour infiltrating lymphocytes were observed in all 50 cases. 20(40%) cases were in grade 1, 18(36%) cases were in grade 2, 4(8%) cases were in grade 3 and the rest 8(16%) cases has no tumour infiltrating lymphocytes. In grade III TIL cases, only one were in stage III compared to 2 in stage I. In no TIL cases, 4 were in stage III disease compared to 2 in Stage I disease. It shows higher TIL is associated with lower stage though conclusion can not be made from this small sample size. Lymphovascular invasion was present in 16(32%) cases and absent in 34 (68%) cases. Ekem et al (2008) found lymphovascular invasion in 33% cases. Out of 16 positive cases 12(75%%) cases had stage III disease and 3(19%) cases had stage II and one (6%) case in stage IV disease, which indicates higher stage disease in lymphovascular invasion positive tumour. As stage is the major factor in predicting prognosis (Rosai, 2004). In this study, majority of the cases in stage III disease show lymphovascular invasion. This partly explains the importance of reporting lymphovascular invasion and Tumour infiltrating lymphocytes in surgical pathology report. Percentage of signet ring cells in the tumour tissue were recorded. 17(34%) cases show 1- 49% signet ring cells and 33 (66%) cases with no signet ring cells. No case having more than 50% signet ring cells was present in this series. 58
  • 68. Out of 50 colorectal cancer cases circumferential margin were involved in 12 (24%) of the cases. Of twelve involved cases 8 cases were in stage III and 4 cases were in stage II disease. Perineural invasion was present in 15(30%) cases. Out of 15 cases 10 cases had stage III disease, 3 cases had stage II disease and one case each in stage I and stage IV. Tumour border configuration were examined microscopically. Infiltrative pattern was seen in 26 (52%) cases and pushing pattern were seen in 24(48%) cases. In 26 infiltrative pattern cases, 15 cases in stage III, 6 cases in stage II, 4 cases in stage I and one case in stage IV. This study shows higher stage disease in circumferential margin involved, perinural invasive and tumour with infiltrative border cases. Rosai (2004) commented circumferential margin, perineural invasion and infiltrative tumour border are adverse prognostic factors in colorectal cancer (CRC). TNM Staging (Turner, 2010) of colorectal cancer was done in all the cases. The maximum number of cases 22 (44%) were in stage III, 14 (28%) were in stage II and 13 (26%) in stage I and one (2%) in stage IV. This study shows only 26% stage 1 cases which indicates delay in diagnosis of the colorectal cancer cases. Derwinger et al (2010) found 41% cases in stage III, 36% cases in stage II, 12% cases in stage IV and 10% cases in stage I. In this present study 49 colon cancer specimens were accompanied by lymph nodes. Of these 23(47%) had nodal metastasis. It indicates patients are in advanced stage at the time of attending physician. In 23 nodal metastasis cases 17(74%) were moderately differentiated and 6(26%) cases were poorly differentiated tumour. 59
  • 69. In addition to environmental factors, genetic and epigenetic mechanisms (DNA methylation) are involved in the pathogenesis of Colorectal carcinoma (Turner, 2010). DNA methylation abnormalities is such important epigenetic changes in CRC. Methylation abnormalities are also observed in other tumours like stomach , pancrease, breast, lung cancers (Samarakoon, 2010). In this study DNA methylation changes was anlyzed in 24 cases of CRC tumour tissue and corresponding normal colonic tissue. This study shows methylation difference between the tumour and normal colonic tissue. A number gene loci are hypermethylated compared to normal tissue and a number of gene loci are hypomethylated compared to normal tissue (Figure 4.8). This study also shows the factors responsible for methylation changes in tumour tissue. Methylation difference is observed between individual cases, location of tumour (right colon tumour tissue is compared with left colon tumour tissue) and between male and female tumour tissue. Methylation difference was also observed between poorly differentiated and moderately differentiated adenocarcinoma (Figure 4.10). Some of the candidate genes showing hypo or hypermethylation is also identified and shown in table 4.11 in result observation section. Kim et al (2010) also found in their study that GATA4 gene is 70% methylated in CRC compared to only 6% methylated in normal colonic tissue and ITGA4 gene is 92% methylated in CRC compared to 13% methylated in normal tissue. 60
  • 70. The exact role of DNA methylation in molecular pathogenesis of cancer is not fully understood. It needs further study to conclue the role of DNA methylation in the pathogenesis of colorectal carcinoma. 61
  • 71. 6.0 Summary and conclusion 6.1 Summary This study was carried out to find histomorphological features, selected clinical data in 50 colonic cancer cases and DNA methylation changes in 24 of these cases. In this study the mean age was 47±14.8 years and 58% of the cases were below the age 50 years which was 10 years less than that described by western observers. Male cases appears to be slightly more than females. Clinical features and related investigations are more or less similar that of other countries. Recent trend show right shift (more cancers in the right side than left sided colon) of CRC in western countries but is is not the findings in this study. Adenocarcinoma of usual pattern was most common type, other common type was mucin secreting adenocarcinoma. 78% cases were moderately differentiated and 22% cases were poorly differentiated. No well differentiated cases were observed in this series indicates higher grade tumours in this study subjects. Most common stage was stage III (44%) cases and 24% cases were in stage I. Lymphnode metastasis was present in 47% cases which indicates delay in diagnosis and advanced stage of disease. DNA methylation changes was seen in tumour tissue compared to normal tissue. Other factors for methylation changes were location of the tumour, differentiation of the tumour and case to case variation. Some genes were identified which are methylated in colorectal cancer tissue but needs further study with large sample size to specify the association.
  • 72. 6.2 Conclusion Histomorphological and selected clinical data presented in this study are more or less similar to that in other published reports, with the exception of mean age of the cases which is about one decade less than western population. DNA methylation changes are observed in the tumour tissue. Some genes are also identified but needs further study to confirm the association and their role in pathogenesis.
  • 73. 7.0 BIBLIOGRAPHY Afroza, A, Hasan, S, Rukunuzzaman, M, Hussain, SA, and Amin, R 2007, ‘Carcinoma- Rectum in an 11 Years old Boy’, Mymensingh Medical Journal, 16(2 Suppl), 70- 72. Aljebreen, AM 2007, ‘Clinico-Pathological Patterns of Colorectal Cancer in Saudi Arabia: Younger with an Advanced Stage Presentation’, The Saudi Journal of Gastroenterology, 33, 84-87. Ayyub, MI, Alradi, AO, Khazeinder, AM, Nagi, AH and Maniyar, IA 2002, ‘Clinicopathological trends in colorectal cancer in a tertiary care hospital’, Saudi Medical Journal, 23, 160-163. Borley, NR 2008, ‘The large intestine’ in Gray's Anatomy, 40th edition, Elsevier company, Pheladelphia, USA, 1137-1160. Boyle, P and Langman, JS 2000, ‘ ABC of colorectal cancer: Epidemiology’, British Medical Journal, 321, 805-808. Cooper, H. S (ed) 1999, ‘Intestinal Neoplasm’ in Diagnostic Surgical Pathology, 3rd edition, Sternberg, S. S. Lippincott Williams and Wilkins, Philadelphia, 2, 1413- 1461. Derwinger, K, Kodeda, K, Bexe-Lindskog, E And Taflin, H 2010, ‘Tumour differentiation grade is associated with TNM staging and the risk of node metastasis in colorectal cancer’, Acta Oncologica, 49, 57–62. Ekem, TE, Bahadir, B, Gun, BD, Bektas, S, Kertis, G, Yurdakan, G and Ozdamar, SO 2008, ‘Colorectal carcinomas: Clinicopathologic investigation, correlation with expression of estrogen and progesterone receptors’, Turkish Journal of Cancer, 38, 118-122.
  • 74. Fireman, Z, Neiman, E, Mouch, SA And Kopelman, Y 2005, ‘Trends in incidence of colorectal cancer in Jewish and Arab population in central Israel’, Digestion, 72, 223-227. Ganong, W.F.(ed) 2005, ‘ Regulation of Gastrointestinal function’ in Review of Medical Physiology, 22nd edition, The Mcgraw-hill companies, New York. pp 479-513. Gomez, D, Dalai, Z, Raw, E, Roberts, C and Lyndon, PJ 2004, ‘Anatomical distribution of colorectal cancer over 10 year period in a district general hospital: is there a true rightward shift?’, Postgraduate Medical Journal, 80, 667-669. Hamilton, SR, Vogelstein, B, Kudo, S, Riboli, E, Nakamura, S, Hainaut, P, Rubio, CA, Sobin, L H, Fogt, F, Winawer, SJ, Goldgar, DE and Jass, JR 2000, ‘Carcinoma of the colon and rectum’ in Pathology and Genetics of Tumour of the Digestive System, Hamilton S.R. and Aaltonen, L. A, IARC press, Lyon, France, pp103-142. Herman, JG and Baylin, SB 2003, ‘Gene Silencing in Cancer in Association with Promoter Hypermethylation’, The New England Journal of Medicine, 349, 2042-54. Hossain, T 2007, ‘Clinicopathological study of colorectal carcinoma’. MD Thesis, Bangabandhu Sheikh Mujib Medical University. Junquira, LC, Carneiro, J and Kelly, RO 1995, Basic Histology, 9th edition, Lange Medical Publishers, California, 397-402. Keating, J, Pater. P, Lolohea, S and Wickremesekera, K 2003, ‘The epidemiology of colorectal cancer: what can we learn from the New Zealand Cancer Registry’, The New Zealand Medical Journal, 116, 1-8.
  • 75. Kim, SK, Lee, J and Sidransky, D 2010, ‘DNA methylation markers in colorectal cancer’, Cancer Metastasis Review,29, 181-206. Leon, MPD, Marino, M, Benatti. P, Rossi, G, Menegatti, M, Pedron, M, Gregorio, C D, Losi, L, Borghi, F, Scarsclli, A, Ponti, G, Roncari, B, Zangardi, G, Abbati, G, Ascari, E, and Roncucci, L 2004, ‘ Trend of incidence, sub site distribution and staging of colorectal neoplasms in the 15-year experience of a specialized cancer registry’, Annals of Oncology, 15, 940-946. Notani, PN 2001, ‘Global variation in cancer incidence and mortality’, Current Science, 81, 467-468. Ogino, S, Kawasaki, T, Nosho, K, Ohnishi, M, Suemoto, Y, Kirkner, GJ and Fuchs, CS 2008, ‘LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer’, International Journal of Cancer, 122, 2767–2773 Riddell, RH, Petras, RE, Williams, GT and Sobin, LH 2003, ‘Epithelial neoplasia of the intestines’ in Tumors of the Intestines , Third series,Fascicle 32, Armed forces institute of pathology,Washington,DC, pp85-240. Ropponen, KM, Eskelenen, MJ, Lipponen, PK, Alhava, E and Kosma, VM 1997, ‘Prognostic Value of Tumour Infiltrating Lymphocytes (TIL) in Colorectal Cancer’, Journal of Pathology,182, 318-324. Rosai, J.(ed) 2004, ‘Gastrointestinal tract’ in Ackerman's Surgical Pathology, 9th edition, Mosby company, St Luis, 1, 776-825. Sadler, T.W.(ed), 1995. Langman's Medical Embryology, 7th edition, Willams and Wilkins, Baltimore, 329-331.
  • 76. Samarakoon, PS 2010, ‘ Epigenomics and Genome wide Methylation Profiling’, Sri Lanka Journal of Bio-Medical Informatics , 1, 53-62. Turner, JR 2010, ‘The Gastrointestinal Tract’ in Robbins and Cotran Pathologic Basis of Disease, 8th edition, Kumar, V. Abbas, A.K. Fausto, N and Aster, J. C . W B Saunders company, Philadelphia,Pennsylvania, 822-825. Wong, JJL, Hawkins, NJ and Ward, RL 2007, ‘Colorectal cancer: a model for epigenetic tumorigenesis’, Gut, 56, 140-148. Yawe, KT, Bakari, AA, Pindiga, UH and Mayun, AA 2007, ‘Clinicopathological pattern and challenges in the management of colorectal cancer in sub-Sahar Africa’, Journal of Chinese Clinical Medicine, 2, 688-694. Young, B and Heath, JW 2000, ‘Gastrointestinal tract’ in Fundamental Histopathology, 4th edition, Harcout publishers Ltd, Churchill livingstone, pp 249-273.
  • 77. Acknowledgements I convey my respect and gratitude to my teacher honorable professor Dr. Mohammed Kamal, Chairman, Department of Pathology, Bangabandhu Sheikh Mujib Medical University (BSMMU), Dhaka and supervisor of this study for his guidance, valuable suggestions, constant supervision, affectionate advice and whole hearted co-operation in making this study a success. Without his vigilance and care this research would have been difficult. I would like to express my sincere gratitude to my teacher Professor (Dr.) Ashim Ranjan Barua for his valuable suggestions and co-operation. I would pay my deep gratitude to my teacher Professor (Dr.) Kamrul Hasan Khan for his valuable advice and co-operation. I am thankful to my teacher Dr. Tamanna Choudhury, Associate professor, Department of Pathology, BSMMU, Dhaka for her valuable suggestions and co-operation. I am greatly thankful to Professor Pran Gopal Datta, Vice-Chancellor, BSMMU, Dhaka for providing me an opportunity to perform this study in this University. I am grateful to my teacher and co-supervisor, Dr. Ferdousy Begum, Assistant Professor, Department of Pathology, BSMMU, for her advice, meticulour correction and active participation in this study. I am highly grateful to my teachers Dr. AKM Nurul Kabir, Dr. Sultana Gulshana Banu, Dr. Suraiya Enam, Dr. Saequa Habib and Dr. Shabnom Akhter, Assistant Professors, Department of Pathology, BSMMU for their help and co-operation.
  • 78. I must thank and pay my gratitudes to DR. Muhammad G. Kibriya and Dr. Farzana Jasmine, Research associate- Assistant Professors, Department of Health Studies (BSD), The University of Chicago Medical Center, Chicago , IL , USA, for their constant encouragement, support, suggestions and immediate response to my e-mails regarding this study. I am very grateful to Prof. Zahidul Haq, Professor, Department of Surgery, BSMMU for his help, co-operation and suggestions for collecting specimen for this study. I would like to extend my sincere thanks to all my colleagues specially Dr. Himel, Dr. Tahera, Dr. Nizam, Dr. Babul, Dr. Taufiq, Dr. Rupash, Dr. Alamgir, Dr. Shahadat and all other residents for their advice, sympathy and co-operation during my study. I would like to appreciate Mr. Osman Goni Chowdhury, Mr. Ruhi Das Roy and other staff of the Department of Pathology, BSMMU for their help and co-operation. I am thankful to Mr. Shelabrata Barua (Jewel) and Md. Faruk hossain who took the trouble to computer compose and typing this thesis. I acknowledge all patients involved in this study and their relatives for co-operation and providing me valuable informations that were helpful in the research work. I wish to express my highest regards and heartiest gratitude to my beloved parents Md. Muzibur Rahman Sarkar and Mrs. Khaleda Begum who always wanted me to be a good and honest teacher. My two younger brothers who always supported me and help me to take any desision. And also my students who inspired me to be a good teacher and researcher. All credit belongs to almighty Allah.
  • 79. Septeber, 2010 Dr. Abu Khalid Muhammad Maruf Raza
  • 81. APPENDIX-II Clinical proforma Title: Study on pathological aspects of colorectal carcinoma. Identification: 1.Case number- 2.Lab number- 3.Date- 4.Name- 5.Age- 6.Sex- 7.Adress- 8.Occupation- Presenting complaints: Tumor Location: Family history: Socio-economic history: Relevent investigation: Haemoglobin- ESR- CEA- Others- Gross examination: Specimen type- Tumor type- Tumor configuration- Tumor size- Mesorectum- Microscopic examination: ii
  • 82. Histologic type- Histologic grade- Extent of invasion- Margins- Proximal- Distal- Circumferential- Lymphovascular invasion- Perinural invasion- Tumour infiltrating lymphocytes- Tumor border configuration- Signet ring cells- Extracellular mucin- Additional pathologic findings- Diagnosis: Consultant pathologist: iii
  • 83. APPENDIX -III PREPARATION OF STAINS AND CHEMICALS: HAEMATOXYLIN AND EOSIN STAINS: a) Harris’ Haematoxylin: Haematoxylin crystal 5.00 gm Absolute alcohol 50.ml Ammonium alum 100.00 gm Distilled water 950. ml Mercuric oxide 2.50 gm Glacial acetic acid 40.00 ml Procedure: Haematoxylin crystals were dissolved in alcohol at 56 C in an oven. Alum was also dissolved by heating. These two solutions were mixed thoroughly and brought to boil rapidly. Then it was removed from the flame and mercuric oxide was added slowly. The solution was then allowed to cool rapidly in cold water. The solution was ready to use after cooling. It was kept in room temperature and filtered before use. 2-4 ml of glacial acetic acid was added per 100 ml of solution to enhance the precision of nuclear stain. b) Acid alcohol: Hydrochloric acid (pure) 1 ml 70% ethyl alcohol (mixed thoroughly) 99 ml c) Eosin solution: Eosin (water soluble) 1gm Distilled water 100 ml Procedure : Eosin dissolved in distilled water and mixed thoroughly. It was stored at room temperature and filtered before use. Solution was made more intense by adding glacial acetic acid in the proportion of 0.2 ml for 100 ml solution before use. iv
  • 84. APPENDIX-IV TISSUE PROCESSING AND STAINING PROCEDURE Tissue processing was done in Department of Pathology, BSMMU by standard protocol in automatic tissue processor (BAVIMED 2050, BAVIMED Laborgeneratebau GmBH, Birkeau, GERMANY). Processed tissue than properly embedded on melted paraffin for making blocks and sections. The sections were stained with haematoxylin and eosin for microscopic examination. Paraffin embedding: Metallic moulds were used for this purpose. These moulds were first lubricated with liquid paraffin. Then melted paraffin was poured into it. The tissue was carefully embedded in proper plane at the bottom of the mould. The respective number of the tissue was inserted into the paraffin wax by the side of the mould. The melted paraffin was then allowed to harden at room temperature. After hardening, the mould was removed and blocks were trimmed properly to mount on a block holder. Then the blocks were kept in ice chamber of a refrigerator for sometime before cutting the sections. Section cutting: Each block of tissue mounted on the holder was fitted in the microtome machine. The microtome knife was properly sharpened before. A water bath with regulated temperature of 45 to 50 C was used for floatation. Sections were cut at 4-5 micron thickness. Ribbons of good sections were selected and floated on luke warm water in the water bath. The sections were then taken on albuminized glass slides. The slides were kept in inclined position to drain off excess water and allowed to dry at room temperature. Staining: All the slides for histopathological examination were stained by routine Haematoxylin and Eosin method v
  • 85. SPTEPS OF HAEMATOXYLIN AND EOSIN STAINING: a) Deparaffinization : i) Slides were kept in hot air oven 15 minutes ii) Xylol- I 5 minutes iii) Xylol –II 5 minutes b) Hydration : i) Absolute alcohol – I 3 minutes ii) Absolute alcohol –II 2 minutes iii) 95% alcohol 2 minutes iv) 70% alcohol 2 minutes v) 50% alcohol 2 minutes vi) Running tap water 2 minutes c) Staining with Haematoxylin and Eosin: Harris’s Haematoxylin 10 minutes 1% acid alcohol 1 minutes Section were placed in running tap water till the sections become blue. Counter staining was done by immersing the section in 1% watery solutions of eosin for 1 minute. d) Dehydration: The sections after Haematoxylin and Eosin staining were dehydrated in following manner. i) 50% alcohol 02 minutes ii) 70% alcohol 02 minutes iii) 80% alcohole 02 minutes iv) 95% alcohol 02 minutes v) absolute alcohol I 02 minute vi) absolute alcohol II 02 minutes vi
  • 86. e) Clearing: a) Xylol – I - 5 minutes b) Xylol – II - 5 minutes c) Xylol – III - 2 minutes. 6) Mounting: Mounting was done with DPX using No. 1 cover slip. Results: Cell nuclei - Blue Cytoplasm - Pink Collagen fibre - Pink RBC - Bright red vii
  • 87. APPENDIX-V Protocol for DNA extraction and purification from colonic tissue (Gentra Puregene Handbook 04/2010, QIAGEN company, USA (www.qiagen.com). : 1. Dissect tissue sample quickly and freeze in liquid nitrogen. Grind frozen tissue in liquid nitrogen with a mortar and pestle. Work quickly and keep tissue on ice at all times, including when tissue is being weighed. 2. Dispense 300 tube on ice, and add the ground tissue from the previous step. Complete cell lysis by following step 2a or 2b below: 2 a. Heat at 65°C for 15 min to 1 h. 2 b. If maximum yield is required, add ene Proteinase K, mix by inverting 25 times, and incubate at 55°C for 3 h or until tissue has completely lysed. Invert tube periodically during the incubation. The sample can be incubated at 55°C overnight for maximum yields. n, and mix the sample by inverting 25 times. Incubate at 37°C for 15–60 min. 4. Incubate for 1 min on ice to quickly cool the sample. 20 s at high speed. 6. Centrifuge for 3 min at 13,000–16,000 x. The precipitated proteins should form a tight pellet. If the protein pellet is not tight, incubate on ice for 5 min and repeat the centrifugation. and add the supernatant from the previous step by pouring carefully. Be sure the protein pellet is not dislodged during pouring. Glycogen Solution
  • 88. 8. Mix by inverting gently 50 times. 9. Centrifuge for 1 min at 13,000–16,000 x g. 10. Carefully discard the supernatant, and drain the tube by inverting on a clean piece of absorbent paper, taking care that the pellet remains in the tube. pellet. 12. Centrifuge for 1 min at 13,000–16,000 x. 13. Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper, taking care that the pellet remains in the tube. Allow to air dry for 5 min. The pellet might be loose and easily dislodged. Avoid over-drying the DNA pellet, as the DNA will be difficult to dissolve. speed to mix. 15. Incubate at 65°C for 1 h to dissolve the DNA. 16. Incubate at room temperature overnight with gentle shaking. Ensure tube cap is tightly closed to avoid leakage. Samples can then be centrifuged briefly and transferred to a storage tube.
  • 89. APPENDIX-VI Protocol DNA methylation (Infinium Assay Methylation Protocol Guide, Catalog # WG- 901-2701 Part # 11322371 Rev. A,Illumina Inc, USA.) 1. Bisulphite conversion of DNA material: After DNA extraction bisulphite conversion of DNA was done by using the EZ DNA MethylationTM Kit (Zymo Research, Catalogue # 500, USA). The kit is based on the reaction that takes place between unconverted cytosine and sodium bisulfite where cytosine is converted into uracil. With subsequent amplification this converted Uracil becomes T (thimine). So instesd of unmethylated C we get T in the sequence. 1 µg DNA from each tissue sample were used for bisulfite conversion. These bisulphite converted DNA were used immediately for methylation assay. 2. Amplification of DNA: The Bisulfite converted DNA samples were denatured by alkali to open up the strands and then was isothermally amplified using Multi-sample Amplification. Master Mix provided by Illumina by overnight incubation. 3. Fragmentation, precipitation and hybridization of DNA: The amplified product was fragmented by a controlled enzymatic process (FMS reagent provided by Illumina. The process uses end-point fragmentation to avoid overfragmenting the samples. These fragmented DNA was precipitated by adding and spinning with isopropanol. Then the x
  • 90. precipitated DNA was resuspended in hybridization buffer (RA1 reagent provided by Illumina). After resuspension, the fragmented DNA was heat denatured. Then samples were put on methylation bead chip. Twelve samples are applied on to each BeadChip, which keeps them separate with an IntelliHyb seal. The prepared BeadChip is incubated overnight in the Illumina Hybridization Oven at 48° c. 4. Wahing, extension and staining of Beadchips: On the following day unhybridized and non-specifically hybridized DNA were removed by washing, and the chips were prepared for staining. A single base extension reaction of the hybridized methylated or unmethylated sequence was done by incorporating fluor tagged necleotide. 5. Imaging Bead Chip: This was done by using Illumina Bead Array Reader software. Intensity of methylation was calculated by intensity of the methylated sequence(x)/ intensity of the methylated sequence (x) + intensity of the umethylated sequence (y). If both the alleles are methylated, intensity = x/x+0=1 and if both the alleles are unmethylated then intensity = 0/0+y=0 and if one allele is methylated and the other is unmethylated then intensity is=x/x+y. xi
  • 91. Appendix - VII American Joint Committee on Cancer (AJCC) TNM Classification of Colorectal Carcinoma (Turner, 2010). TUMOUR Tis In situ dysplasia or intramucosal carcinoma T1 Tumor invades submucosa T2 Tumor invades into, but not through, muscularis propria T3 Tumor invades through muscularis propria T3a Invasion <0.1 cm beyond muscularis propria T3b Invasion 0.1 to 0.5 cm beyond muscularis propria T3c Invasion >0.5 to 1.5 cm beyond muscularis propria T3d Invasion >1.5 cm beyond muscularis propria T4 Tumor invades adjacent organs or visceral peritoneum T4a Invasion into other organs or structures T4b Invasion into visceral peritoneum REGIONAL LYMPH NODES NX Lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastasis in one to three regional lymph nodes N2 Metastasis in four or more regional lymph nodes DISTANT METASTASIS MX Distant metastasis cannot be assessed M0 No distant metastasis M1 Distant metastasis or seeding of abdominal organs
  • 92. TNM staging for colorectal carcinoma (Turner, 2010) Stage-I Tl N0 M0 or T2, N0, M0 Stage-II T3 N0 M0 or T4, N0, M0 Stage-III T any Nl M0 or T any,N2,N3, M0 Stage-IV T any N any Ml
  • 93. Appendix–VIII MasterTable Sl.No. CaseID Lab.No. Age/Sex C/F Hb(mg/dl) CEA (ng/ml) Location Size(Cm) Diagnosis Differentia tion TILgrade Ext.Muc Signetring cell LV invasion PN invasion Tumour border Circumfer ential margin LN/Inv Growth pattern Tumour stage 1C1B-7750/0935/Fp/r/b11.18.01Rec6AdeM2NoNoPrePInfInv11/11UlIII 2C2B-114-16/1019/Mp/r/b10.83.14Rec4AdeMNNoNoAPInfInv7/0ExoII 3C3KA-119-2375/Mp/r/b11672Rec3AdeMNNoNoAAPushInv7/3UlIII 4C4B-174/1068/Mpain1110.9Cae10MucP1>50%1-49%AAPushFree10/0ExoII 5C5B-107-8/1041/Mp/r/b and pain 73.9Tran6MucPN>50%1-49%AAPushFree4/0ExoI 6C6B-268/1052/F/p/r/b9.316.8Rec4AdeP1NoNoAAPushFree8/0ExoI 7C7B-273/1055/Mp/r/b11.279.64Rec6MucPN>50%1-49%AAPushInv0/0InfII 8C8B-327/1025/Mp/r/b10.83.9Rec4AdeM1NoNoPrePInfFree7/4ExoIII 9C9B-331/1052/Mpain104.1Asc6AdeM11-49%1-49%PreAInfFree11/1UlcIII 10C10KA-266-6965/Fa/b11.19.3Rec8AdeM11-49%NoAAInfFree11/5ExoIII 11C12B-444/1045/Mp/r/b1318.34Rec5AdeM11-49%NoPreAInfFree4/2UlcIII 12C13KA-399-40245/Mp/r/b9.39.1Rec7AdeM1NoNoAAInfFree4/0UlcII 13C14KA-406-0743/Mp/r/b10.111.1Rec5AdeMNNoNoAAInfFree7/0ExoI 14C15KA423-26-35/FPain1168.9Rec5AdeP11-49%1-49%AAInfFree12/1UlcIII 15C16B-566/1035/Mp/r/b1113.7Rec6AdeM1NoNoPrePInfiltr ative Free7/1ExoIII xiv
  • 94. Appendix–VIII MasterTable Sl.No. CaseID Lab.No. Age/Sex C/F Hb(mg/dl) CEA (ng/ml) Location Size(Cm) Diagnosis Differentia tion TILgrade Ext.Muc Signetring cell LV invasion PN invasion Tumour border Circumfer ential margin LN/Inv Growth pattern Tumour stage 16C17B-589/1024/MPain12.7 0 0.31Cae10MucP1>50%NoAAPushInv20/15UlcIII 17C19B-960/1059/Mp/r/b10.61.9Hepa6AdeP2NoNoAAPushFree20/0ExoII 18C20B-1126/1028/Ma/b10.63.1Rec9AdeM1NoNoAAPushFree14/0ExoI 19C21B-1370/1053/Mp/r/b10.224.6Rec5AdeM21-49%NoAAInfFree7/0ExoI 20C22B-1433/1084/Fmass11.64.44Rec4AdeM11-49%NoAAPushFree11/0UlcI 21C23B-1467/1030/Ma/b123.9Rec7AdeM1No1-49%PrePInfFree19/1UlcIII 22C25B-1706/1027/Fp/r/b and pain 11.13.1Hepa6AdeM11-49%NoprePInfFree23/1UlcIII 23C26KB-705-0848/Fp/r/b123.19Rec4AdeM3NoNoAAPushFree10/0ExoI 24C27B-1851/1036/Mp/r/b93.9Rec10AdeM1NoNoAAInfFree12/0ExoI 25C28B-1995/1028/Fp/r/b13.13.9Rec4AdeM31-49%1-49%preAInfFree4/0UlcII 26C30KC-61- 63/10 59/Fpain11.841.2Rec7AdeM31-49%NoAAInfFree8/0ExoI 27C31BH-779- 80/10 50/Mp/r/b and pain 11.63.9Asc6AdeM21-49%NoAAInfFree4/0ExoII 28C32KC-320-2342/Ma/b131.81Rec3AdeM11-49%1-49%AAPushFree2/0ExoI 29C33B-2397- 99/10 58/Fa/b12.85.5Rec6AdeM1NoNoPrePInfFree15/0ExoII 30C34B-2415- 16/10 37/Mp/r/b14.5314Des5MucPN>50%1-49%PrePInfInvolve d 17/17InfIII xv
  • 95. Appendix–VIII MasterTable Sl.No. CaseID Lab.No. Age/Sex C/F Hb(mg/dl) CEA (ng/ml) Location Size(Cm) Diagnosis Differentia tion TILgrade Ext.Muc Signetring cell LV invasion PN invasion Tumour border Circumfer ential margin LN/Inv Growth pattern Tumour stage 31C35B-2626/1045/Fa/b11.27.49Rec8AdePN1-49%1-49%AAInfFree15/1ExoIII 32C36B-2613/1045/Fa/b12234Des4AdeMNNoNoPrePInfInv25/20UlcIII 33C37KD-236-4140/Fw/p13.13.38Rec5AdeM2No1-49%PreAPushFree9/3UlcIII 34C38KD236-41-73/Mp/r/b and pain 13.524.8Hepa3AdeM2 1-49%1-49% AAPushFree15/0UlcI 35C39B-2888/1050/Fpain5.42.17Sig5AdeP2No1-49%APPushFree16/0UlcII 36C40KD-715-1966/Mp/r/b14.62.1Rec4AdeM1NoNoAPPushFree10/0UlcI 37C41KD-809-1130/Fp/r/b and pain 12.74.1Rec7AdeM2NoNoAAPushFree8/0ExoI 38C42B-2976/1050/Fw/p10.93.75Rec5AdeM21-49%NoAAPushi ng Free7/2UlcIII 39C43B-3376- 79/10 28/Mp/r/b and pain 13.813.57Rec5.5MucP1>50%1-49%AAInfInv5/2UlcIII 40C44B-3374/1052/Mmass8.84.34Rec5AdeM1NoNoAAPushFree15/9UlcIII 41C45KD-868-7150/Ma/b6.91.63Tran4.5AdeM3NoNoAAPushFree4/3ExoIII 42C46B-3395-9663/Fp/r/b12.738.9Rec5AdeM2NoNoAAPushFree5/0UlcII 43C47B-3469/1038/Fa/b1029.3Asc8AdeM2NoNoAPInfFree7/0UlcIII 44C48B-3484- 85/10 50/Mw/p11.6256Asc3AdeM21-49%NoPrePInfFree7/4UlcIV 45C49B-3604- 05/10 48/Fw/p10.75.1Rec4AdeM21-49%NoAAPushFree6/0ExoII xvi
  • 96. Appendix–VIII MasterTable Sl.No. CaseID Lab.No. Age/Sex C/F Hb(mg/dl) CEA (ng/ml) Location Size(Cm) Diagnosis Differentia tion TILgrade Ext.Muc Signetring cell LV invasion PN invasion Tumour border Circumfer ential margin LN/Inv Growth pattern Tumour stage 46C50B-3738- 39/10 65/Mw/p815.9Asc10AdeM21-49%1-49%AApushInv11/0ExoII 47C51B-3906/1039/Fw/p8.61.9Rec6AdeP21-49%1-49%PrePInfInv9/9InfIII 48C52B-4307/1039/Fmass8.11.09Sig5AdeM2NoNoPreAInfFree7/0UlcII 49C53B-4328- 29/10 72/Ma/b12.74.5Rec4AdeM2NoNoAAPushInv5/0UlcII 50C54B-4407/1038/Fw/p632.19Asc6AdeM2NoNopreAPushInv9/1UlcIII M=male,f=female,p/r/b=perectalbleeding,w/p=weakness,pallor,anorexia,mass=abdominalmass,a/b=alteredbowelhabit,Rec=Rectum,Asc=Asendingcolon,Tran=Transeverse colon,Des=descendingcolon,sig=Sigmoidcolon,cae=caecum,Ade=Adenocarcinomausualtype,Muc=Mucinouscarcinoma.P=Present,A=absent,push=pushingmargin,Inf= Infiltratingmargin,Ulc=Ulcerated,Exo=Exophytic,Inf=infiltrative.Inv=Involved,Til=tumourinfiltratinglymphocytes,Ext.Muc=Extracellularmucin,LVinvasion=Lymphovascular invasion,PNinvasion=Perineuralinvasion.LN=lymphnode. xvii