Professional Documents
Culture Documents
DEPARTMENT OF PATHOLOGY
Bangabandhu Sheikh Mujib Medical University
Dhaka, Bangladesh
2010
Contents Page
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1 INTRODUCTION 01
2 REVIEW OF LITERATURE
a Anatomical consideration 04
b Physiology 08
c Tumors of the colon and rectum 09
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
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
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
environmental factors including diatery factors, obesity, alcohol intake, smoking, life style
and genetic and epigenetic abnormalities. The molecular events that leads to CRC is
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
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
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
2
1.1 AIM
To see the morphologic pattern, clinical features and DNA methylation changes in
1.2 OBJECTIVES
3
2.0 REVIEW OF LITERATURE
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
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
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
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
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
The large intestine is divided topographically into three segments ( Junquira et al, 1995):
5
a. Colon,
b. Rectum and
c. Anal canal.
a. Mucosa
b. Submucosa
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
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
Adventitia or serosa consists of thin connective tissue layer covered by the mesothelium
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
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
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
8
2.3 Tumours of the colon and rectum
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
1. Non-neoplastic polyp
i. Hyperplastic polyp
2. Epithelial tumors
1. Adenoma
i. Tubular adenoma
9
2. Intraepithelial neoplasia (Dysplasia) associated with Chronic inflammatory
diseases
3. Carcinoma
i. Adenocarcinoma
v. Adenosquamous carcinoma
4. Carcinoid tumour
5. Mixed carcinoid-adenocarcinoma
6. Non-epithelial tumor
i. Lipoma
ii. Leiomyoma
10
iv. Leiomyosarcoma
v. Angiosarcoma
7. Secondary tumors
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
between 60 and 79 years. Fewer than 20% cases occur before the age of 50 years (Turner,
2010).
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,
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
(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
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
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
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
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.
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
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 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
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
Most of the colon cancer is adenocarcinoma. In the study of Keating et al, (2003)
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
2.3.3 Pathogenesis
Genetic susceptibility may be the soil on which subsequent environmental factors act
(Riddell, 2003).
Epidemiologic studies have indicated that meat consumption, smoking, sedentary lifestyle
and alcohol consumption are risk factors for colorectal carcinoma. Inverse associations
and includes genetic and epigenetic abnormalities (Turner, 2010). The genetic and
2. Loss of p53.
3. K-ras mutation.
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
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
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
16
2.3.3.4 DNA methylation in colorectal cancer:
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
Hypermethylated genes are associated with colorectal neoplasia includes the tumour
suppressor, mismatch-repair and cell-cycle regulatory genes (Wong, 2007). Table 2.3.1
colorectal cancer.
17
Table: 2.3.1 Genes silenced by hypermethylation in colorectal cancer (Wong, 2007)
Genes Function
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
lead to loss of imprinting (LOI) which can drive cellular proliferation in cancer. The
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
19
2.3.5 Microscopic features of colorectal carcinoma
characterized by good gland formation and varying degrees of differentiation, from well to
have 5-50% glandular structure and undifferentiated carcinoma have <5% glandular
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
2.3.5.3 Signet ring cell adenocarcinoma: Signet-ring and mucinous cell carcinoma
definied by the presence of >50% of the tumour cells with prominant intracytoplasmic
2.3.5.4 Small cell carcinoma: A rare variant is small cell carcinoma which composes <1%
squamous carcinoma and adenocarcinoma either as separate areas within the tumour or
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
malignant cells with vesicular nuclei, prominent nucleoli and abundant pink cytoplasm
(Hamilton, 2000).
physician regarding patient's prognosis and the need for adjuvant therapy. For many years,
and the TNM classification(Turner, 2010). Table 2.3.2 shows TNM classification and
21
Table:2.3.2 TNM Classification of Colorectal Carcinoma by American Joint
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)
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).
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
23
Age: Tumour occuring in very young and old patient have poor prognosis.
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.
Vascular invasion: When venous invasion is present, the 5-years survival decreases
markedly.
Surgical margins: Presence of tumour in surgical margin is the single most critical factor
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
HLA-DR expression: Patient with tumour having strong HLA-DR expression show better
prognosis.
25
3. MATERIAL AND METHOD
a. The clinical and histopathological part of this study was carried out at the
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
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
26
3.4 Exclusion criteria
b. Non Hodgkin lymphoma and other non epithelial tumors of the colon.
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
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.
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
27
a. Recording of gross features of the specimen:
ii. Tumour characteristics: Tumor size including thickness, extent around bowel,
invasion of adjacent organs and distance of the tumour from the proximal and
iii. Number of lymph nodes found and size of the largest node.
All the tissue blocks were submitted for routine processing and paraffin
Embedding (appendix-III). The tissue processing and staining was performed following
sections stained with haematoxyline and eosin staining method were carried out during
28
3.6.3 Routine microscopic examination
Routinely stained sections examined first under low power and then under high power
- Adenocarcinoma(NOS).
- Mucinous.
- Small cell.
- Well differentiated.
- Moderately differentiated.
- Poorly differentiated.
-Submucosa.
-Muscle coat.
-Serosa.
-Pericolic fat.
- Pushing.
29
- Infiltrative.
x. Lymphocytic response
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
Paired unfixed tissue samples, one each from tumour and healthy mucosa were taken in
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
DNA extraction were done using Gentra Purgene Tissue kit (Qiagen Catalogue #
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
Histopathological portion: All the necessary and relevant data were recorded
methodically and meticulously as far as possible in the clinical proforma. Relevant data
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
data were recorded. DNA methylation of non tumorous areas of colon of the same patient
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
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.
33
Figure 4.1 : Bar diagram showing age distribution of colorectal cancer.
Number of cases
Age in decades
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.
34
Table 4.1 : Sex distribution in relation to age group.
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.
35
Table 4.2: Symptoms with tumour location of colorectal cancer cases.
Haemoglobin levels were recorded in 50 cases. It ranged from 8.3 to 14.6 gm/dl.
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
36
Table 4.3: Haemoglobin concentration in 50 cases of colorectal cancer patients
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
37
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
Caecum 2 2(4%) -
38
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.
3%
Ulcerating
Exophytic
44% 50%
Infiltrative
were male and 21 (42%) cases were female. All 6 (12%) cases of mucinous
39
Table 4.6: Histological diagnosis of 50 cases of colorectal cancer
Adenocarcinoma 44 (88%) 23 21
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 cases, 22 cases were seen in male and 17 cases in female and of 11 poorly
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
40
Table 4.7: Histological grading of colorectal cancer cases with sex and location:
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
41
Table 4.8: TNM (Tumour, Node, Metastasis) stage with sex and location
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.
42
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
8% 16%
Nil
Grade-1
36%
Grade-2
40% Grade-3
43
4.7.5: Lymphovascular invasion in colorectal cancer patients
Lymphovascular invasion was present in 16(32%) cases and absent in 34 (68%) cases
32%
Present
68% Absent
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.
44
Figure 4.5 Extracellular mucin in colorectal cancer patient.
12%
1-49%
52% No Extracellular
Extracellular
36% mucin
mucin
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
1-49% 17(34%)
>50% -
45
4.7.9: Circumferential margin, perinural invasion and tumour border
(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.
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
46
Table 4.10: Circumferential margin involvement, Perineural invasion and
loci. In figure 4.6 scatter diagram shows a linear correlation between the total intensity
was seen which indicates there is no bias in the microarray intensity data. Figure 4.6
47
Figure 4.6: Scatter diagram showing total intensity in tumour tissue and its
corresponding normal tissue in all 24 cases
vs Index
50000
40000
Tumour Intensity
30000
20000
10000
There was significant difference in the methylation status between tumour and sorrounding
normal tissue.
Average beta is the methylation status of a gene locus. It is calculated by intensity of the
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
48
hypermethylated. The central line represents regression line and other two lines on either
vs Index
Tumour
1
0.80
Tumour AVG_Beta
0.60
0.40
0.20
0 Normal
0 0.20 0.40 0.60 0.80 1
Normal AVG_Beta
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
49
Figure 4.8: Histogram showing tumour differential score.
12000
10000
# of Occurrences
8000
6000
4000
2000
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
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
50
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
51
Figure 4.10 Variation of methylation data due to tumour differentiation.
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
52
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
53
5.0 DISCUSSION
In this study, 50 cases of colorectal cancer were analyzed to find out histomorphological
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.
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
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
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
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
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.
In Among 37 cases of left colon cancer cases, 33 cases were in rectum. This shows higher
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
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
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,
This partly explains the importance of reporting lymphovascular invasion and Tumour
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
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
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
59
In addition to environmental factors, genetic and epigenetic mechanisms (DNA
Methylation abnormalities are also observed in other tumours like stomach , pancrease,
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.
colon tumour tissue is compared with left colon tumour tissue) and between male and
female tumour tissue. Methylation difference was also observed between poorly
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%
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
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
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
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
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.
63
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
63
7.0 BIBLIOGRAPHY
Rectum in an 11 Years old Boy’, Mymensingh Medical Journal, 16(2 Suppl), 70-
72.
Ayyub, MI, Alradi, AO, Khazeinder, AM, Nagi, AH and Maniyar, IA 2002,
Borley, NR 2008, ‘The large intestine’ in Gray's Anatomy, 40th edition, Elsevier company,
1461.
colorectal cancer in Jewish and Arab population in central Israel’, Digestion, 72,
223-227.
of colorectal cancer over 10 year period in a district general hospital: is there a true
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.
Junquira, LC, Carneiro, J and Kelly, RO 1995, Basic Histology, 9th edition, Lange Medical
colorectal cancer: what can we learn from the New Zealand Cancer Registry’,
65
Kim, SK, Lee, J and Sidransky, D 2010, ‘DNA methylation markers in colorectal cancer’,
Ascari, E, and Roncucci, L 2004, ‘ Trend of incidence, sub site distribution and
Notani, PN 2001, ‘Global variation in cancer incidence and mortality’, Current Science,
81, 467-468.
Riddell, RH, Petras, RE, Williams, GT and Sobin, LH 2003, ‘Epithelial neoplasia of the
Ropponen, KM, Eskelenen, MJ, Lipponen, PK, Alhava, E and Kosma, VM 1997,
Rosai, J.(ed) 2004, ‘Gastrointestinal tract’ in Ackerman's Surgical Pathology, 9th edition,
Sadler, T.W.(ed), 1995. Langman's Medical Embryology, 7th edition, Willams and
Turner, JR 2010, ‘The Gastrointestinal Tract’ in Robbins and Cotran Pathologic Basis of
Wong, JJL, Hawkins, NJ and Ward, RL 2007, ‘Colorectal cancer: a model for epigenetic
Yawe, KT, Bakari, AA, Pindiga, UH and Mayun, AA 2007, ‘Clinicopathological pattern
67
Acknowledgements
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.
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:
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
a) Harris’ Haematoxylin:
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:
c) Eosin solution:
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
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
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
a) Deparaffinization :
b) Hydration :
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:
following manner.
vi
e) Clearing:
a) Xylol – I - 5 minutes
b) Xylol – II - 5 minutes
c) Xylol – III - 2 minutes.
6) Mounting:
Results:
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 μl Cell Lysis Solution into a 1.5 ml grinder tube on ice, and add the
ground tissue from the previous step. Complete cell lysis by following step 2a or 2b below:
3. Add 1.5 μl RNase A Solution, and mix the sample by inverting 25 times. Incubate at
37°C for 15–60 min.
5. Add 100 μl Protein Precipitation Solution, and vortex vigorously for 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.
7. Pipet 300 μl isopropanol into a clean 1.5 ml microcentrifuge tube and add the
supernatant from the previous step by pouring carefully. Be sure the protein pellet is not
dislodged during pouring. If the DNA yield is expected to be low (<1 μg) add 0.5 μl
Glycogen Solution
viii
8. Mix by inverting gently 50 times.
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.
11. Add 300 μl of 70% ethanol and invert several times to wash the DNA pellet.
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
14. Add 200 μl DNA Hydration Solution and vortex for 5 s at medium speed to mix.
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.
ix
APPENDIX-VI
Protocol DNA methylation (Infinium Assay Methylation Protocol Guide, Catalog # WG-
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.
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
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
necleotide.
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
xi
Appendix - VII
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
DISTANT METASTASIS
MX Distant metastasis cannot be assessed
M0 No distant metastasis
M1 Distant metastasis or seeding of abdominal organs
xii
TNM staging for colorectal carcinoma (Turner, 2010)
xiii
Appendix – VIII
Master Table
Hb (mg/dl)
Differentia
Signet ring
Circumfer
Size (Cm)
TIL grade
Diagnosis
Ext. Muc
Tumour
LN/ Inv
Age/ Sex
margin
border
Location
Lab. No.
invasion
invasion
ential
Case ID
Tumour
Growth
tion
C/F
(ng/ml)
Sl. No.
pattern
stage
CEA
PN
cell
LV
1 C1 B-7750/09 35/F p/r/b 11.1 8.01 Rec 6 Ade M 2 No No Pre P Inf Inv 11/11 Ul III
2 C2 B-114-16/10 19/M p/r/b 10.8 3.14 Rec 4 Ade M N No No A P Inf Inv 7/0 Exo II
3 C3 KA-119-23 75/M p/r/b 11 672 Rec 3 Ade M N No No A A Push Inv 7/3 Ul III
4 C4 B-174/10 68/M pain 11 10.9 Cae 10 Muc P 1 >50% 1-49% A A Push Free 10/0 Exo II
5 C5 B-107-8/10 41/M p/r/b 7 3.9 Tran 6 Muc P N >50% 1-49% A A Push Free 4/0 Exo I
and
pain
6 C6 B-268/10 52/F/ p/r/b 9.3 16.8 Rec 4 Ade P 1 No No A A Push Free 8/0 Exo I
7 C7 B-273/10 55/M p/r/b 11.2 79.64 Rec 6 Muc P N >50% 1-49% A A Push Inv 0/0 Inf II
8 C8 B-327/10 25/M p/r/b 10.8 3.9 Rec 4 Ade M 1 No No Pre P Inf Free 7/4 Exo III
9 C9 B-331/10 52/M pain 10 4.1 Asc 6 Ade M 1 1-49% 1-49% Pre A Inf Free 11/1 Ulc III
10 C10 KA-266-69 65/F a/b 11.1 9.3 Rec 8 Ade M 1 1-49% No A A Inf Free 11/5 Exo III
11 C12 B-444/10 45/M p/r/b 13 18.34 Rec 5 Ade M 1 1-49% No Pre A Inf Free 4/2 Ulc III
12 C13 KA-399-402 45/M p/r/b 9.3 9.1 Rec 7 Ade M 1 No No A A Inf Free 4/0 Ulc II
13 C14 KA-406-07 43/M p/r/b 10.1 11.1 Rec 5 Ade M N No No A A Inf Free 7/0 Exo I
14 C15 KA423-26- 35/F Pain 11 68.9 Rec 5 Ade P 1 1-49% 1-49% A A Inf Free 12/1 Ulc III
15 C16 B-566/10 35/M p/r/b 11 13.7 Rec 6 Ade M 1 No No Pre P Infiltr Free 7/1 Exo III
ative
xiv
Appendix – VIII
Master Table
Hb (mg/dl)
Differentia
Signet ring
Circumfer
Size (Cm)
TIL grade
Diagnosis
Ext. Muc
Tumour
LN/ Inv
Age/ Sex
margin
border
Location
Lab. No.
invasion
invasion
ential
Case ID
Tumour
Growth
tion
C/F
(ng/ml)
Sl. No.
pattern
stage
CEA
PN
cell
LV
16 C17 B-589/10 24/M Pain 12.7 0.31 Cae 10 Muc P 1 >50% No A A Push Inv 20/15 Ulc III
0
17 C19 B-960/10 59/M p/r/b 10.6 1.9 Hepa 6 Ade P 2 No No A A Push Free 20/0 Exo II
18 C20 B-1126/10 28/M a/b 10.6 3.1 Rec 9 Ade M 1 No No A A Push Free 14/0 Exo I
19 C21 B-1370/10 53/M p/r/b 10.2 24.6 Rec 5 Ade M 2 1-49% No A A Inf Free 7/0 Exo I
20 C22 B-1433/10 84/F mass 11.6 4.44 Rec 4 Ade M 1 1-49% No A A Push Free 11/0 Ulc I
21 C23 B-1467/10 30/M a/b 12 3.9 Rec 7 Ade M 1 No 1-49% Pre P Inf Free 19/1 Ulc III
22 C25 B-1706/10 27/F p/r/b 11.1 3.1 Hepa 6 Ade M 1 1-49% No pre P Inf Free 23/1 Ulc III
and
pain
23 C26 KB-705-08 48/F p/r/b 12 3.19 Rec 4 Ade M 3 No No A A Push Free 10/0 Exo I
24 C27 B-1851/10 36/M p/r/b 9 3.9 Rec 10 Ade M 1 No No A A Inf Free 12/0 Exo I
25 C28 B-1995/10 28/F p/r/b 13.1 3.9 Rec 4 Ade M 3 1-49% 1-49% pre A Inf Free 4/0 Ulc II
26 C30 KC-61- 59/F pain 11.8 41.2 Rec 7 Ade M 3 1-49% No A A Inf Free 8/0 Exo I
63/10
27 C31 BH-779- 50/M p/r/b 11.6 3.9 Asc 6 Ade M 2 1-49% No A A Inf Free 4/0 Exo II
80/10 and
pain
28 C32 KC-320-23 42/M a/b 13 1.81 Rec 3 Ade M 1 1-49% 1-49% A A Push Free 2/0 Exo I
29 C33 B-2397- 58/F a/b 12.8 5.5 Rec 6 Ade M 1 No No Pre P Inf Free 15/0 Exo II
99/10
30 C34 B-2415- 37/M p/r/b 14.5 314 Des 5 Muc P N >50% 1-49% Pre P Inf Involve 17/17 Inf III
16/10 d
xv
Appendix – VIII
Master Table
Hb (mg/dl)
Differentia
Signet ring
Circumfer
Size (Cm)
TIL grade
Diagnosis
Ext. Muc
Tumour
LN/ Inv
Age/ Sex
margin
border
Location
Lab. No.
invasion
invasion
ential
Case ID
Tumour
Growth
tion
C/F
(ng/ml)
Sl. No.
pattern
stage
CEA
PN
cell
LV
31 C35 B-2626/10 45/F a/b 11.2 7.49 Rec 8 Ade P N 1-49% 1-49% A A Inf Free 15/1 Exo III
32 C36 B-2613/10 45/F a/b 12 234 Des 4 Ade M N No No Pre P Inf Inv 25/20 Ulc III
33 C37 KD-236-41 40/F w/p 13.1 3.38 Rec 5 Ade M 2 No 1-49% Pre A Push Free 9/3 Ulc III
34 C38 KD236-41- 73/M p/r/b 13.5 24.8 Hepa 3 Ade M 2 A A Push Free 15/0 Ulc I
and 1-49% 1-49%
pain
35 C39 B-2888/10 50/F pain 5.4 2.17 Sig 5 Ade P 2 No 1-49% A P Push Free 16/0 Ulc II
36 C40 KD-715-19 66/M p/r/b 14.6 2.1 Rec 4 Ade M 1 No No A P Push Free 10/0 Ulc I
37 C41 KD-809-11 30/F p/r/b 12.7 4.1 Rec 7 Ade M 2 No No A A Push Free 8/0 Exo I
and
pain
38 C42 B-2976/10 50/F w/p 10.9 3.75 Rec 5 Ade M 2 1-49% No A A Pushi Free 7/2 Ulc III
ng
39 C43 B-3376- 28/M p/r/b 13.8 13.57 Rec 5.5 Muc P 1 >50% 1-49% A A Inf Inv 5/2 Ulc III
79/10 and
pain
40 C44 B-3374/10 52/M mass 8.8 4.34 Rec 5 Ade M 1 No No A A Push Free 15/9 Ulc III
41 C45 KD-868-71 50/M a/b 6.9 1.63 Tran 4.5 Ade M 3 No No A A Push Free 4/3 Exo III
42 C46 B-3395-96 63/F p/r/b 12.7 38.9 Rec 5 Ade M 2 No No A A Push Free 5/0 Ulc II
43 C47 B-3469/10 38/F a/b 10 29.3 Asc 8 Ade M 2 No No A P Inf Free 7/0 Ulc III
44 C48 B-3484- 50/M w/p 11.6 256 Asc 3 Ade M 2 1-49% No Pre P Inf Free 7/4 Ulc IV
85/10
45 C49 B-3604- 48/F w/p 10.7 5.1 Rec 4 Ade M 2 1-49% No A A Push Free 6/0 Exo II
05/10
xvi
Appendix – VIII
Master Table
Hb (mg/dl)
Differentia
Signet ring
Circumfer
Size (Cm)
TIL grade
Diagnosis
Ext. Muc
Tumour
LN/ Inv
Age/ Sex
margin
border
Location
Lab. No.
invasion
invasion
ential
Case ID
Tumour
Growth
tion
C/F
(ng/ml)
Sl. No.
pattern
stage
CEA
PN
cell
LV
46 C50 B-3738- 65/M w/p 8 15.9 Asc 10 Ade M 2 1-49% 1-49% A A push Inv 11/0 Exo II
39/10
47 C51 B-3906/10 39/F w/p 8.6 1.9 Rec 6 Ade P 2 1-49% 1-49% Pre P Inf Inv 9/9 Inf III
48 C52 B-4307/10 39/F mass 8.1 1.09 Sig 5 Ade M 2 No No Pre A Inf Free 7/0 Ulc II
49 C53 B-4328- 72/M a/b 12.7 4.5 Rec 4 Ade M 2 No No A A Push Inv 5/0 Ulc II
29/10
50 C54 B-4407/10 38/F w/p 6 32.19 Asc 6 Ade M 2 No No pre A Push Inv 9/1 Ulc III
M=male, f= female, p/r/b= per ectal bleeding, w/p= weakness, pallor, anorexia, mass= abdominal mass, a/b= altered bowel habit, Rec= Rectum, Asc= Asending colon, Tran= Transeverse
colon, Des= descending colon, sig= Sigmoid colon, cae= caecum, Ade= Adenocarcinoma usual type, Muc= Mucinous carcinoma. P= Present, A= absent, push= pushing margin, Inf=
Infiltrating margin, Ulc= Ulcerated, Exo= Exophytic, Inf= infiltrative. Inv= Involved, Til =tumour infiltrating lymphocytes, Ext. Muc= Extra cellular mucin, LV invasion= Lymphovascular
invasion, PN invasion= Perineural invasion. LN= lymphnode.
xvii