Laboratory Investigation of Colorectal Cancer

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Title: Discuss the laboratory investigation of colorectal cancer. Refer to the pathophysiology, treatment and screening in your answer.

The development of cancer from the colon or rectum is given the term colorectal cancer (CRC). CRC usually begins as a tumour that is benign such as a polyp and over a period progresses into cancer. It affects both men and women. Over 2,700 people are diagnosed with CRC every year and it is the second most common cancer in Ireland. If diagnosed early, prognosis is good (Siegel et al., 2011).

Signs and symptoms of colorectal cancer.

Signs and symptoms varying depending on the tumour location in the bowel, and if it has metastasized. The typical warning signs are: bloody stool, constipation that is worsening, reduced stool thickness, decreased appetite, weight loss and vomiting or nauseas in a person aged over 50 years. About 50% of people that have CRC have no symptoms (Siegel et al., 2011).

Etiology of colorectal cancer

More than 75-95% of CRC happens in people with minimal or absent genetic risk. Risk factors are older people, male, high fat diet, red and processed meat, alcohol, smoking, obesity and absent physical exercise. About 10% of CRCs are associated with not enough activity. The risk linked with alcohol seems to rise with more than a single drink a day. Streptococcus gallolyticus is linked with CRC (Siegel et al., 2011).

Individuals with inflammatory bowel disease, such as Crohn’s disease and ulcerative colitis, have a higher risk of CRC. The longer the person has the disease the higher the risk and the more severe the disease. Prevention using aspirin and frequent colonoscopies in these high-risk groups are recommended. Less than 2% of CRCs diagnosed yearly, inflammatory bowel disease accounts for (Siegel et al., 2011).

People with a history in the family with two or more first degree relatives have a two or three- fold increase of developing CRC and this category of people account for approximately 20% of cases. Some genetic syndromes are also linked with an increased risk of developing CRC. Hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) is the most common one, which is seen in approximately 3% of cases of CRC. Other syndromes which are strongly linked with CRC are familial adenomatous polyposis (FAP) and Gardner syndrome. CRC almost always occurs in these people and accounts for 1% of CRC cases. Most deaths as a result of CRC are linked with the metastasis of the disease. Metastasis association in colon cancer 1 (MACC1) is a gene that seems to add to the ability for the metastasis of the disease. This gene encodes a transcriptional factor which influences the hepatocyte growth factor expression. Epigenetic factors like abnormal tumour suppressor promoter and DNA methylation play a part in the progression of CRC (Verhulst et al., 2012).

Pathogenesis of Colorectal cancer.

CRC originates from epithelial cells which are lining the colon or the rectum in the gastrointestinal trac, most often because of mutations of the Wnt signaling pathway which raises the signaling activity. These mutations may be acquired or inherited, and probably happen in the intestinal crypt stem cell. The APC gene is the most often gene that is mutated in all CRC, which creates the APC protein. The APC protein stops the build-up of the protein β-catenin. In the absence of APC, β-catenin builds up to high amounts and translocate to the nucleus, where it binds to DNA and the transcription of oncogenes is activated. Normally, these genes are vital for renewal of stem cells and their differentiation, but if inappropriately produced at high amounts, this can lead to cancer. Some cancers have higher β-catenin as a result of β-catenin (TNNB1) mutations which stops its own destruction or there are mutations in other genes that function similarly to APC like AXIN1, AXIN2, NKD1 or TCF7L2 (Verhulst et al., 2012).

Outside the Wnt signaling pathway defects, additional mutations must happen for the cell to develop into cancer. The TP53 gene that produces the protein P53, generally regulates cell mitosis and destroys cells if they have defects in the Wnt pathway. The cell line eventually gets a mutation in the P53 gene and this transforms the tissue from benign to malignant. The gene sometimes encoding the P53 protein isn’t mutated, but instead another protein that is protective termed BAX is instead mutated (Verhulst et al., 2012).

There are other proteins such as DCC (Deleted in CRC) and TGF-β, often deactivated in CRC are responsible for programmed cell death. Sometimes a downstream protein termed SMAD is deactivated instead of TGF-β. A deleted chromosome segment is common in DCC in CRC (Verhulst et al., 2012).

About 70% of all genes are normally expressed in CRC with about 1% that are over expressed in CRC in contrast to other cancers. The order of which mutation occur is sometimes important. If previously an APC mutation happened, a primary KRAS mutation normally develops to cancer. A tumour suppressor PTEN generally stops PI3K but sometimes can get mutated and become deactivated (Kang et al., 2005).

It has been found that CRC can be grouped revealed by genome scale analysis, into non-hypermutated and hypermutated tumour types. As well as the inactivating and the oncogenic mutations, also contained in the non-hypermutated samples are mutated FAM123B, CTNNB1, SOX9, ARID1A and ATM. Hypermutated tumours form developing through a specific group of genetic events demonstrate forms of TGFBR2, ACVR2A, MSH3, MSH6, TCF7L2, SLC9A9 and BRAF. What is common among these genes and across both tumor types, their connection with the TGF-β and Wnt signaling pathway which causes increased MYC activity, is a key player in CRC (Kang et al., 2005).

Deficient mismatch repair (MMR) tumours are defined by an MMR protein deficiency which are generally as a result of inherited mutations and or epigenetic silencing. 15 to 18% of CRC tumours are MMR deficient, with 3% developing because of Lynch syndrome. The mismatch repair system role is to protect the genetic material integrity in cells. Therefore, an MMR deficiency can result in an inability to sense and fix damage to genetic material, giving way to more mutations causing cancer and allowing progression of CRC (Kang et al., 2005).

The classical model of CRC pathogenesis is the polyp to cancer progression sequence. This is phases of transition from a tumour that is benign to CRC over a period of many years. Gene mutations, alterations in epigenetics and changes in local inflammation are central to the polyp to CRC progression (Kang et al., 2005).

Diagnosis of colorectal cancer

CRC is diagnosed by performing a biopsy of the area of the colon suspected of possible development of tumours, usually in a sigmoidoscopy or colonoscopy, depending on the lesion location. Microscopical assessment of the biopsy is used to confirm diagnosis (Leopoldo et al., 2008).

A CT scan of the abdomen, chest and pelvis is used to determine the presence of metastasis. The cancer stage is based on both pathological and radiological results. Tumour staging is based on the TNM system where it considers how much the primary tumour has spread and if there are metastases into the lymph nodes and distant organs (Leopoldo et al., 2008).

Macroscopy analysis

Tumours that develop on the right side of the colon (caecum and ascending colon) tend to grow outwards. They rarely cause obstruction. Tumours that develop from the left side tend to be circumferential and may cause obstruction (Leopoldo et al., 2008).

Microscopic analysis

Adenocarcinoma is a type of CRC that is a malignant epithelial type tumour, that arises from the superficial glandular epithelial cells that are lining the rectum and colon. This tumour invades the colon wall, infiltrating the muscularis mucosae, the submucosa layer and eventually, the muscularis propria. The microscopic appearance of the tumour cells is tubular structures that are irregular, contain pluristratification, multiple lumens and reduced amount of stroma (cells back to back). Tumour cells sometimes secrete mucus which can invade the interstitium creating large mucus pools. This is present in mucus adenocarcinoma, where the cells are poorly differentiated. If the mucus stays in the cell, it moves the nucleus to the cell periphery, this happens in signet ring cell. Adenocarcinoma can present in three different degrees of differentiation depending on the glandular architecture, cellular pleomorphism and mucosecretion of the main pattern: well, moderately or poorly (Leopoldo et al., 2008).



Figure 1: Haematoxylin and eosin stain if colon tissue with moderately differentiated adenocarcinoma demonstrating complicated glandular structures in a desmoplastic stroma (Li, 2005).


Figure 2: Haematoxylinand eosin stain of mucinous adenocarcinoma demonstrating extracellular mucin (Li, 2005).

Figure 3: signet ring cell carcinoma (Li, 2005).

Immunohistochemistry:

Immunohistochemistry is used to ascertain correct diagnosis in cases where a metastasis from

CRC is suspected. Some proteins can be used as diagnostic markers such as CK20 and MUC2 as

they are more specifically expressed in CRC. Lynch syndrome a genetic disorder with increased

risk of CRC and other cancers can be screened for using immunohistochemistry. Specific genetic

mutations in genes MLH1, MSH2, MSH6 and PMS2 occur in Lynch syndrome and its diagnosis

can be made by looking for these specific mutations. To guide treatment and assist in determining

the prognosis, immunohistochemistry can also be used (Hinoi et al., 2001).


Figure 4: Immunohistochemistry of a MSI tumour demonstrating

(A): Loss MLH1 expression. (B): Normal MSH2 expression. (C): Normal MSH6 expression. 

(D): Loss of PMS2 expression (Li, 2005).

Micro-satellite Instability testing

Impaired DNA mismatch repair (MMR) can result in genetic hyper-mutability (predisposition to mutation) which is called micro-satellite instability (MSI). Phenotypic evidence that MMR is not functioning normally indicates the presence of MSI. During DNA replication, errors that occur spontaneously, such as short insertions, single base mismatches or deletions, MMR corrects them. Polymerase errors are corrected by proteins involved in MMR by creating a complex which attaches to the mismatch area of DNA, removes the error and replaces it with the correct sequence (Hinoi et al., 2001).

Errors that occur during DNA replication in cells with abnormally functioning MMR are unable to correct these errors and consequently accumulate errors. The creation of novel micro-satellite fragments is caused by this. These novel microsatellites can be revealed using polymerase chain reaction-based assays and provide evidence for the presence of MSI. Repeated sequences of DNA are microsatellites (Hinoi et al., 2001).

Associated with CRCs as well as other cancers are microsatellite instability (MSI). CRC is the most prevalent in MSI. MSI can be divided into MSI-Low (MSI-L), MSI-High (MSI-H) or Microsatellite stable (MSS). More positive prognosis is with MSI-H than MSI-L or MSS tumours. CRC with less metastasis is associated with MSI-H than the other two. Lynch syndrome is indicated when MSI-H is positive in a tumour and a non-metastasis prognosis and less aggressive treatment required (Hinoi et al., 2001).

Amsterdam criteria II

Is a diagnostic set of criteria which doctors use to aid in identifying families that are more than likely to develop Lynch syndrome, also referred to as hereditary non-polyposis CRC (HNPCC), the Amsterdam criteria is used to diagnose for.

The following criteria each of which must be fulfilled:

  1. Three or more relations with a linked cancer.
  2. Two or more previous generations with the cancer
  3. One or more relations diagnosed before the age of fifty years
  4. Familial adenomatous polyposis (FAP) should be left out in cases of CRC.
  5. Pathologic examination should verify the tumours (Vasen, 2007).

Bethesda guidelines

These guidelines are less stringent. These are used to identify families with the likelihood of having MMR gene mutations.

  1. CRC or uterine cancer diagnosed in a person before the age of 50 years
  2. The presence of metachronous, synchronous CRC or other HNPCC linked tumours, in the absence of age.
  3. CRC with MSI-H, histopathology diagnosed in a person who is under 60 years of age.
  4. CRC is diagnosed in at least one or greater, first degree relatives, with HNPCC associated tumour, with one of the cancers diagnosed under 50 years old.
  5. CRC diagnosed in two or more first or second-degree relatives with HNPCC associated tumours, in the absence of age (Vasen, 2007).

BRAF testing

This test helps to distinguish between HNPCC and sporadic cancer by testing for the V600E mutation in the BRAF gene. The hypermethylation in the MLH1 gene promoter may result in the loss of the MLH1 protein expression in the sporadic cancer. Tumours are suspected of being associated with HNPCC or Lynch syndrome if there is a loss of the MLH1 protein but neither BRAF V600E mutations nor hypermethylation (Vasen, 2007).

Prevention of CRC

About half of CRC cases it has been estimated are due to lifestyle factors and around a quarter of all CRC cases are preventable. Engaging in physical activity, increasing surveillance, eating a diet high in fiber and cutting back on smoking and intake of alcohol lowers the risk (Vasen, 2007).

Screening

Greater than 80% of CRCs begin from adenomatous polyps, screening for this type of cancer is found effective for both early detection of the cancer and for preventing it. CRC diagnosis through screening tend to happen 2-3 years prior to diagnosis of cases that have symptoms. Polyps that are detected may be removed, normally by sigmoidoscopy or colonoscopy and therefore, stop them from developing into cancer. Screening can lower CRC death by about 60% (BowelScreen, 2019).

Faecal occult blood testing, colonoscopy and flexible sigmoidoscopy are the three primary tests used in screening (BowelScreen, 2019).

Faecal occult blood testing (FOBT) of a faecal sample is normally recommended every two years and may either be immunochemical, or guaiac based. If the results of the FOBT are abnormal, the individual is normally sent for a colonoscopy. This FOBT screening lowers CRC deaths by about 16% yearly to every two years and those participating in the screening may be reduced by up to 23% (BowelScreen, 2019).

The national Bowel screening program in Ireland is BowelScreen. This screening program provides a free home test to women and men who are aged from 60 to 69 years every 2 years. It is easy and quick to use test that is non-invasive and can be done at home. The home test called FIT which means faecal immunochemical test, which detects for traces of blood in the stool which are not visible by the naked eye (BowelScreen, 2019).

Treatment of colorectal cancer

The aim of treatment in CRC is cure or palliation. Various factors are considered when deciding which aim to adopt such as the individual’s health and their preference, also the stage the tumour is at. Surgery can be curative if CRC is detected early. Although, if caught at a later stage, where it has metastasized, it is less likely and treatment therefore is aim at palliation, to relieve the symptoms the tumour is causing and to make the individual as comfortable as possible (Lea, Allingham-Hawkins and Levine, 2010).

Surgery

If at an early stage CRC is found, it can be removed in a colonoscopy. For those with localised cancer, complete surgical removal of the tumour with complete margins is the preferred treatment, with the goal of achieving a cure. An open laparotomy or sometimes laproscopically, can be used Lea, Allingham-Hawkins and Levine, 2010).

Chemotherapy

In both colon and rectal cancer, in certain cases chemotherapy can be used in addition to surgery. Depending on the stage of the disease, the decision will be based on whether to add chemotherapy in the management of the cancer. No chemotherapy is offered in stage I and surgery are the definitive treatment. The role of chemotherapy is debatable in stage II and not normally offered unless certain risk factors like undifferentiated tumour, T4 tumour, vascular and perivascular invasion or insufficient lymph node sampling is identified. Chemotherapy is an integral part in stage III and IV (Weisenberger et al., 2006).

Radiation therapy

While it may be useful a combination of chemotherapy and radiation in rectal cancer, its use is not routine in colon cancer because of the sensitivity of the colon to radiation. For some stages of rectal cancer, radiation therapy may be used in a neoadjuvant and adjuvant setting (Weisenberger et al., 2006).

Immunotherapy

It has found to be useful immunotherapy with immune checkpoint inhibitors for a particular type of CRC that has mismatch repair deficiency and microsatellite instability (Weisenberger et al., 2006).

Palliative care

It is recommended palliative care for any individual who has advanced CRC or has significant symptoms. What can be consisted of in palliative care is procedures that relieve symptoms or complications from the cancer but does not try to cure the underlying cancer, therefore improving their quality of life (Weisenberger et al., 2006).

Conclusion

CRC is a disease that can be prevented. If caught early such as through screening programmes, there is excellent prognosis. It is a complex disease with genetic alterations that allows the progression of a benign polyp overtime into a malignant tumour, which is the classical model of the development of CRC. The important gene mutations involved in CRC are the APC gene, KRAS oncogene, the mismatch repair of DNA leading to mutations in MLH1, MSH2, MSH6, PMS1 and PMS2 genes. The detection of these mutated genes has become a way in diagnosing the type of CRC such as HNPCC. The treatment of choice for CRC depends on the stage and the metastasis of the tumour, which surgery the preferred if the cancer is localized.

References:

  • BowelScreen (2019). BowelScreen – The National Bowel Screening Programme of Ireland. [online] Bowelscreen.ie. Available at: https://www.bowelscreen.ie/ [Accessed 30 Jan. 2019].
  • Hinoi, T., Tani, M., Lucas, P., Caca, K., Dunn, R., Macri, E., Loda, M., Appelman, H., Cho, K. and Fearon, E. (2001). Loss of CDX2 Expression and Microsatellite Instability Are Prominent Features of Large Cell Minimally Differentiated Carcinomas of the Colon. The American Journal of Pathology, 159(6), pp.2239-2248.
  • Kang, H., O’Connell, J., Maggard, M., Sack, J. and Ko, C. (2005). A 10-Year Outcomes Evaluation of Mucinous and Signet-Ring Cell Carcinoma of the Colon and Rectum. Diseases of the Colon & Rectum, 48(6), pp.1161-1168.
  • Lea, A., Allingham-Hawkins, D. and Levine, S. (2010). BRAF p.Val600Glu (V600E) Testing for Assessment of Treatment Options in Metastatic Colorectal Cancer. PLoS Currents, 2, p.RRN1187.
  • Leopoldo, S., Lorena, B., Cinzia, A., Gabriella, D., Angela Luciana, B., Renato, C., Antonio, M., Carlo, S., Cristina, P., Stefano, C., Maurizio, T., Luigi, R. and Cesare, B. (2008). Two Subtypes of Mucinous Adenocarcinoma of The Colorectum: Clinicopathological and Genetic Features. Annals of Surgical Oncology, 15(5), pp.1429-1439.
  • Li, Z. (2005). Tumor angiogenesis and dynamic CT in colorectal carcinoma: Radiologic-pathologic correlation. World Journal of Gastroenterology, 11(9), p.1287.
  • Siegel, R., Ward, E., Brawley, O. and Jemal, A. (2011). Cancer statistics, 2011. CA: A Cancer Journal for Clinicians, 61(4), pp.212-236.
  • Vasen, H. (2007). Review article: the Lynch syndrome (hereditary nonpolyposis colorectal cancer). Alimentary Pharmacology & Therapeutics, 26, pp.113-126.
  • Verhulst, J., Ferdinande, L., Demetter, P. and Ceelen, W. (2012). Mucinous subtype as prognostic factor in colorectal cancer: a systematic review and meta-analysis. Journal of Clinical Pathology, 65(5), pp.381-388.
  • Weisenberger, D., Siegmund, K., Campan, M., Young, J., Long, T., Faasse, M., Kang, G., Widschwendter, M., Weener, D., Buchanan, D., Koh, H., Simms, L., Barker, M., Leggett, B., Levine, J., Kim, M., French, A., Thibodeau, S., Jass, J., Haile, R. and Laird, P. (2006). CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nature Genetics, 38(7), pp.787-793.
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