Clinicopathologic pattern and DNA methylation status of colorectal carcinoma in a group of Bangladeshi patients

Inhaltsverzeichnis

1 INTRODUCTION

2 REVIEW OF LITERATURE
a Anatomical consideration
b Physiology
c Tumors of the colon and rectum

3 MATERIALS AND METHODS

4 OBSERVATIONS AND RESULTS

5 DISCUSSION

6 SUMMARY AND CONCLUSION

7 BIBLIOGRAPHY

8 APPENDICES
I Certificate from The Chicago University
II Clinical proforma
III Preparation of stains and chemicals
IV Tissue processing and staining procedures
V Protocol for DNA extraction
VI Protocol for DNA methylation
VII TNM staging system
VIII Master table
IX Illustrations

LIST OF TABLE

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LIST OF FIGURES

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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 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.

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.

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).

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):

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).

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).

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

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
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 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.

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 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 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).

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.

Table: 2.3.1 Genes silenced by hypermethylation in colorectal cancer (Wong, 2007)

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2.3.3.4. 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).

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).

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