Development of drug resistance is one of the major causes of cancer treatment failure. For a cytotoxic agent to reach a cancer cell, the drug must enter the bloodstream, be activated or escape inactivation by drug-metabolising enzymes, and arrive at the target in its active form. The drug–target interaction must then produce cell death. Resistance to cytotoxic therapy results from interference with one or more of these critical steps. Some of the factors affecting these steps are poor absorption or distribution of the drug; metabolism of the drug in the liver; decreased blood supply to the target organ; tumour microenvironment such as hypoxia; drug target modifications; and mechanisms that block the intracellular accumulation of the drug.
Multidrug resistance refers to the clinical and laboratory circumstances in which a tumour is no longer susceptible to several cytotoxic agents with different mechanisms or targets. P-glycoprotein is a member of the adenosine triphosphate (ATP)-binding receptor family. It spans the plasma membrane and recognises a broad spectrum of cytotoxic drugs. In the presence of ATP, P-glycoprotein pumps the drugs to the extracellular space so that an effective concentration at the intracellular target is never achieved.
2. Answer E
p53 is a tumour suppressor which plays a critical role in the maintenance of genomic integrity; hence its popular designation as ‘guardian of the genome’ (Meek, 2009). The p53 protein is normally expressed at low levels in all cells. However, genetic injuries, such as those that occur through ionising radiation, trigger the stabilisation and activation of p53 protein. p53 functions as a transcription factor, directing expression of p21, an inhibitor of the cyclin-dependent kinases that regulate the cell cycle. Activation of p53 leads to arrest in the G1 phase of the cell cycle, enabling cells to repair DNA damage before proceeding into S phase and DNA replication. In other cells, activation of p53 causes activation of multiple effectors, leading to apoptosis. Mutations of p53 are common in human cancers, being demonstrable in about 50% of cases, and increased levels of its negative regulators MDM2 and MDM4 (also known as MDMX) downregulate p53 function in many of the rest. Most mutations are amino acid substitutions within the DNA-binding domain of p53, resulting in its misfolding and binding to heat shock proteins. The rate of protein turnover is greatly slowed for these mutant p53 molecules. This explains the paradox that high levels of p53 protein in tumour specimens are commonly taken as evidence of a mutation in p53. A germline p53 mutation is associated with a familial syndrome of breast cancer, sarcomas and other neoplasms, known as Li–Fraumeni syndrome.
p53 is a tumour suppressor which plays a critical role in the maintenance of genomic integrity; hence its popular designation as ‘guardian of the genome’ (Meek, 2009). The p53 protein is normally expressed at low levels in all cells. However, genetic injuries, such as those that occur through ionising radiation, trigger the stabilisation and activation of p53 protein. p53 functions as a transcription factor, directing expression of p21, an inhibitor of the cyclin-dependent kinases that regulate the cell cycle. Activation of p53 leads to arrest in the G1 phase of the cell cycle, enabling cells to repair DNA damage before proceeding into S phase and DNA replication. In other cells, activation of p53 causes activation of multiple effectors, leading to apoptosis. Mutations of p53 are common in human cancers, being demonstrable in about 50% of cases, and increased levels of its negative regulators MDM2 and MDM4 (also known as MDMX) downregulate p53 function in many of the rest. Most mutations are amino acid substitutions within the DNA-binding domain of p53, resulting in its misfolding and binding to heat shock proteins. The rate of protein turnover is greatly slowed for these mutant p53 molecules. This explains the paradox that high levels of p53 protein in tumour specimens are commonly taken as evidence of a mutation in p53. A germline p53 mutation is associated with a familial syndrome of breast cancer, sarcomas and other neoplasms, known as Li–Fraumeni syndrome.
Meek, D.W. (2009). Tumour suppression by p53: a role for the DNA damage response? Nat Rev Cancer 9, 714–723.
3. Answer C
Accurately tracking tumour position is critically important when maximising radiation dose to the tumour and limiting normal tissue exposure. In certain locations in the body, such as the lungs, stomach and liver, tumours can move as the patient breathes. In the past, this movement has confounded the ability to precisely map the tumour location and to accurately deliver radiation therapy.
Gating is a system that tracks a patient’s normal respiratory cycle with an infrared camera and chest/abdomen marker (Ahmad et al., 2012). The system is coordinated to only deliver radiation when the tumour is in the treatment field. This prevents unnecessary radiation exposure to normal structures. Lung cancers move with respiration and if this variation is great, four-dimensional CT can be used to obtain a series of CT scans at different phases of the respiratory cycle. The information can help define the motion of the tumour, which can then be targeted with respiratory gating. This involves tracking the patient’s respiratory cycle, commonly using surface markers and delivering treatment at specific phases of the cycle.
Accurately tracking tumour position is critically important when maximising radiation dose to the tumour and limiting normal tissue exposure. In certain locations in the body, such as the lungs, stomach and liver, tumours can move as the patient breathes. In the past, this movement has confounded the ability to precisely map the tumour location and to accurately deliver radiation therapy.
Gating is a system that tracks a patient’s normal respiratory cycle with an infrared camera and chest/abdomen marker (Ahmad et al., 2012). The system is coordinated to only deliver radiation when the tumour is in the treatment field. This prevents unnecessary radiation exposure to normal structures. Lung cancers move with respiration and if this variation is great, four-dimensional CT can be used to obtain a series of CT scans at different phases of the respiratory cycle. The information can help define the motion of the tumour, which can then be targeted with respiratory gating. This involves tracking the patient’s respiratory cycle, commonly using surface markers and delivering treatment at specific phases of the cycle.
Ahmad, S.S, Duke, S., Jena, R., Williams, M.V., and Burnet, N.G. (2012). Advances in radiotherapy. BMJ 345, e7765.
4. Answer A
Oestrogen receptor positivity is a good prognostic factor in breast cancer (Benson et al., 2009). Lymph node involvement is the most important adverse prognostic feature. Increasing grade of tumour and human epidermal growth factor receptor 2 (HER2) overexpression are also associated with poor prognosis. Inflammatory breast cancer has a poor prognosis. BRCA1 and 2 gene mutations as prognostic indicators have not been established
Two factors emerge as the principal determinants of local recurrence within the conserved breast: margin status and the presence or absence of an extensive in-situ component. Lymphatic invasion and young age (<35 years) are primary predictors for increased risk of ipsilateral breast tumour recurrence. Consistent associations have been recorded for larger tumour size (>2 cm) and higher histological grade, but not for tumour subtype or nodal status. Patterns of mRNA and microRNA expression in tumours are also predictive of outcome.
Oestrogen receptor positivity is a good prognostic factor in breast cancer (Benson et al., 2009). Lymph node involvement is the most important adverse prognostic feature. Increasing grade of tumour and human epidermal growth factor receptor 2 (HER2) overexpression are also associated with poor prognosis. Inflammatory breast cancer has a poor prognosis. BRCA1 and 2 gene mutations as prognostic indicators have not been established
Two factors emerge as the principal determinants of local recurrence within the conserved breast: margin status and the presence or absence of an extensive in-situ component. Lymphatic invasion and young age (<35 years) are primary predictors for increased risk of ipsilateral breast tumour recurrence. Consistent associations have been recorded for larger tumour size (>2 cm) and higher histological grade, but not for tumour subtype or nodal status. Patterns of mRNA and microRNA expression in tumours are also predictive of outcome.
Benson, J.R., Jatoi, I., Keisch, M., Esteva, F.J., Makris, A., and Jordan, V.C. (2009). Early breast cancer. Lancet 373, 1463–1479.
5. Answer D
Renal cell carcinoma (RCC) arises from a complex series of mutation and selection events in cells located within the proximal tubules of nephrons, culminating in cancer cells that acquire characteristics allowing immortalisation, resistance to apoptosis, evasion of immunosurveillance, recruitment of angiogenic factors, and ultimately distant spread. A frequent event during the evolution of clear cell RCC is loss of function of the von Hippel–Lindau (VHL) gene, located on chromosome 3p (Gleadle, 2009). Inheritance of a defective copy of the VHL gene leads to VHL disease and is the most common cause for inherited clear-cell RCC in which a second hit or somatic mutation is presumed to occur in the cancerous cells. In addition, up to 75% of patients with sporadic clear-cell RCC have mutation or silencing by methylation of VHL. The VHL gene functions in several pathways linked to carcinogenesis, most notably the hypoxia-inducible pathway. In the absence of VHL, hypoxia-inducible factor α (HIF) accumulates, leading to production of several growth factors, including vascular endothelial growth factor and platelet-derived growth factor, and enhanced glycolysis. Many non-renal cancers are also characterised by hypoxia, enhanced HIF levels and increased expression of hypoxically-regulated genes that correlate both with tumour aggression and patient outcome.
Renal cell carcinoma (RCC) arises from a complex series of mutation and selection events in cells located within the proximal tubules of nephrons, culminating in cancer cells that acquire characteristics allowing immortalisation, resistance to apoptosis, evasion of immunosurveillance, recruitment of angiogenic factors, and ultimately distant spread. A frequent event during the evolution of clear cell RCC is loss of function of the von Hippel–Lindau (VHL) gene, located on chromosome 3p (Gleadle, 2009). Inheritance of a defective copy of the VHL gene leads to VHL disease and is the most common cause for inherited clear-cell RCC in which a second hit or somatic mutation is presumed to occur in the cancerous cells. In addition, up to 75% of patients with sporadic clear-cell RCC have mutation or silencing by methylation of VHL. The VHL gene functions in several pathways linked to carcinogenesis, most notably the hypoxia-inducible pathway. In the absence of VHL, hypoxia-inducible factor α (HIF) accumulates, leading to production of several growth factors, including vascular endothelial growth factor and platelet-derived growth factor, and enhanced glycolysis. Many non-renal cancers are also characterised by hypoxia, enhanced HIF levels and increased expression of hypoxically-regulated genes that correlate both with tumour aggression and patient outcome.
Gleadle, J.M. (2009). Review article: How cells sense oxygen: lessons from and for the kidney. Nephrology (Carlton) 14, 86–93.
6. Answer F
The loss of genomic stability can drive the development of colorectal cancer by facilitating the acquisition of multiple tumour-associated mutations (Markowitz and Bertagnolli, 2009). Oncogenic mutations of RAS and BRAF, which activate the mitogen-activated protein kinase (MAPK) signalling pathway, occur in 37% and 13% of colorectal cancers, respectively. RAS mutations, principally in KRAS, activate the GTPase activity that signals directly to the RAF proto-oncogene serine/threonine-protein kinase. BRAF mutations signal BRAF serine–threonine kinase activity, which further drives the MAPK signalling cascade. BRAF mutations are detectable even in small polyps, and compared with RAS mutations, they are more common in hyperplastic polyps, serrated adenomas and proximal colon cancers. Patients with numerous and large hyperplastic lesions, a condition termed the hyperplastic polyposis syndrome, have an increased risk of colorectal cancer, with disease progression occurring through an intermediate lesion with a serrated luminal border on histological analysis.
Epidermal growth factor (EGF) is a soluble protein that has trophic effects on intestinal cells. Clinical studies have supported an important role of signalling through the EGF receptor (EGFR) in a subgroup of colorectal cancers. EGFR mediates signalling by activating the MAPK and PI3K signalling cascades. Recent clinical data have shown that advanced colorectal cancer with tumour-promoting mutations of these pathways, including activating mutations in KRAS, BRAF and the p110 subunit of PI3K, do not respond to anti-EGFR therapy.
The loss of genomic stability can drive the development of colorectal cancer by facilitating the acquisition of multiple tumour-associated mutations (Markowitz and Bertagnolli, 2009). Oncogenic mutations of RAS and BRAF, which activate the mitogen-activated protein kinase (MAPK) signalling pathway, occur in 37% and 13% of colorectal cancers, respectively. RAS mutations, principally in KRAS, activate the GTPase activity that signals directly to the RAF proto-oncogene serine/threonine-protein kinase. BRAF mutations signal BRAF serine–threonine kinase activity, which further drives the MAPK signalling cascade. BRAF mutations are detectable even in small polyps, and compared with RAS mutations, they are more common in hyperplastic polyps, serrated adenomas and proximal colon cancers. Patients with numerous and large hyperplastic lesions, a condition termed the hyperplastic polyposis syndrome, have an increased risk of colorectal cancer, with disease progression occurring through an intermediate lesion with a serrated luminal border on histological analysis.
Epidermal growth factor (EGF) is a soluble protein that has trophic effects on intestinal cells. Clinical studies have supported an important role of signalling through the EGF receptor (EGFR) in a subgroup of colorectal cancers. EGFR mediates signalling by activating the MAPK and PI3K signalling cascades. Recent clinical data have shown that advanced colorectal cancer with tumour-promoting mutations of these pathways, including activating mutations in KRAS, BRAF and the p110 subunit of PI3K, do not respond to anti-EGFR therapy.
Markowitz, S.D. and Bertagnolli, M.M. (2009). Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med 361, 2449–2460.
7. Answer D
The DNA damage response (DDR) coordinates the repair of DNA and the activation of cell-cycle checkpoints to arrest the cell to allow time for repair. DNA is subject to a high level of endogenous damage and the DDR is essential for the maintenance of genomic stability and survival. Dysregulation of the DDR can lead to genomic instability that promotes cancer development, but this is exploitable with both conventional cytotoxic therapy and DDR inhibitors. Downregulated DDR pathways render the tumour sensitive to specific cytotoxic drugs and some DDR inhibitors. Upregulated DDR pathways confer therapeutic resistance. Inhibitors of the DDR have been developed to overcome resistance and to augment the activity of conventional therapy. Loss of a DDR pathway can lead to dependence on a compensatory pathway, and targeting this second pathway may render endogenous DNA damage cytotoxic by a process termed synthetic lethality, which is tumour-specific because the normal tissues have functional DNA repair.
Mismatch repair (MMR) corrects replication errors that cause the incorporation of the wrong nucleotide (a mismatch) and nucleotide deletions and insertions. Defective MMR increases mutation rates up to 1000-fold, results in microsatellite instability (MSI) and is associated with cancer development.
The DNA damage response (DDR) coordinates the repair of DNA and the activation of cell-cycle checkpoints to arrest the cell to allow time for repair. DNA is subject to a high level of endogenous damage and the DDR is essential for the maintenance of genomic stability and survival. Dysregulation of the DDR can lead to genomic instability that promotes cancer development, but this is exploitable with both conventional cytotoxic therapy and DDR inhibitors. Downregulated DDR pathways render the tumour sensitive to specific cytotoxic drugs and some DDR inhibitors. Upregulated DDR pathways confer therapeutic resistance. Inhibitors of the DDR have been developed to overcome resistance and to augment the activity of conventional therapy. Loss of a DDR pathway can lead to dependence on a compensatory pathway, and targeting this second pathway may render endogenous DNA damage cytotoxic by a process termed synthetic lethality, which is tumour-specific because the normal tissues have functional DNA repair.
Mismatch repair (MMR) corrects replication errors that cause the incorporation of the wrong nucleotide (a mismatch) and nucleotide deletions and insertions. Defective MMR increases mutation rates up to 1000-fold, results in microsatellite instability (MSI) and is associated with cancer development.
8. Answer C
Nucleotide excision repair (NER) removes helix-distorting adducts on DNA, e.g. those caused by ultraviolet (UV) radiation and tobacco smoke, and contributes to the repair of intrastrand and interstrand cross-links (ICLs); the xerodermapigmentosum (XP) proteins and DNA excision repair protein 1 (ERCC1) also have crucial roles in both the NER and ICL repair pathways. ERCC1 polymorphisms are associated with skin and lung cancer. Hereditary defects in NER cause UV sensitivity and skin cancer. Deficiency in NER confers sensitivity to platinum agent therapy, which reflects a reduced capacity to repair ICLs, and the measurement of levels of crucial NER enzymes could thus be used for patient stratification.
Nucleotide excision repair (NER) removes helix-distorting adducts on DNA, e.g. those caused by ultraviolet (UV) radiation and tobacco smoke, and contributes to the repair of intrastrand and interstrand cross-links (ICLs); the xerodermapigmentosum (XP) proteins and DNA excision repair protein 1 (ERCC1) also have crucial roles in both the NER and ICL repair pathways. ERCC1 polymorphisms are associated with skin and lung cancer. Hereditary defects in NER cause UV sensitivity and skin cancer. Deficiency in NER confers sensitivity to platinum agent therapy, which reflects a reduced capacity to repair ICLs, and the measurement of levels of crucial NER enzymes could thus be used for patient stratification.
For Questions 7 and 8:
Curtin, N.J. (2012). DNA repair dysregulation from cancer driver to therapeutic target. Nat Rev Cancer 12, 801–817.
Clinical
9. Answer A
Bevacizumab is a humanised monoclonal antibody that targets vascular endothelial growth factor (VEGF) (Kohne and Lenz, 2009). It has a high binding affinity to VEGF and interferes with binding of VEGF-A to the VEGF receptors VEGFR-1 and VEGFR-2, thus inhibiting VEGF-mediated intracellular signalling. Through inactivation of the VEGF–VEGFR pathway, this targeted agent can help prevent the formation of new blood vessels and tumour growth. It has been shown to improve survival in metastatic colorectal carcinoma when used in combination with chemotherapy. Bevacizumab is generally well tolerated when combined with other drugs, such as irinotecan. Hypertension, the only consistently observed bevacizumab-associated side effect that requires treatment, is manageable with standard oral anti-hypertensives (e.g. calcium-channel blockers, angiotensin converting enzyme inhibitors and diuretics).
Bevacizumab is a humanised monoclonal antibody that targets vascular endothelial growth factor (VEGF) (Kohne and Lenz, 2009). It has a high binding affinity to VEGF and interferes with binding of VEGF-A to the VEGF receptors VEGFR-1 and VEGFR-2, thus inhibiting VEGF-mediated intracellular signalling. Through inactivation of the VEGF–VEGFR pathway, this targeted agent can help prevent the formation of new blood vessels and tumour growth. It has been shown to improve survival in metastatic colorectal carcinoma when used in combination with chemotherapy. Bevacizumab is generally well tolerated when combined with other drugs, such as irinotecan. Hypertension, the only consistently observed bevacizumab-associated side effect that requires treatment, is manageable with standard oral anti-hypertensives (e.g. calcium-channel blockers, angiotensin converting enzyme inhibitors and diuretics).
Kohne, C.H. and Lenz, H.J. (2009). Chemotherapy with targeted agents for the treatment of metastatic colorectal cancer. Oncologist 14, 478–488.
10. Answer D
The clinical picture is one of carcinoid syndrome secondary to a carcinoid tumour (Kulke and Mayer, 1999). The syndrome of facial flushing, watery diarrhoea, abdominal pain and cardiac abnormalities imply hepatic metastases of a carcinoid tumour. Common sites of carcinoid tumour in the bowel are the terminal ileum, rectum and appendix (25% of all tumours). Diagnosis is made from the clinical picture and 24-h collection of urine for levels of 5-HIAA, a metabolite of 5-HT (serotonin) that is postulated to be responsible for the cardiac manifestations of the disease and profuse watery diarrhoea.
The 24-h urine collection for the presence of VMA (metabolites of catecholamines) may be helpful in making the diagnosis of phaeochromocytoma, a rare catecholamine-producing tumour. In adults, 90% of tumours are in the adrenal medulla; 90% of these tumours are benign and 80% are unilateral.
Somatostatin is a peptide that inhibits hormone production by several tumours and its analogue octreotide is useful in the treatment of carcinoid syndrome.
The clinical picture is one of carcinoid syndrome secondary to a carcinoid tumour (Kulke and Mayer, 1999). The syndrome of facial flushing, watery diarrhoea, abdominal pain and cardiac abnormalities imply hepatic metastases of a carcinoid tumour. Common sites of carcinoid tumour in the bowel are the terminal ileum, rectum and appendix (25% of all tumours). Diagnosis is made from the clinical picture and 24-h collection of urine for levels of 5-HIAA, a metabolite of 5-HT (serotonin) that is postulated to be responsible for the cardiac manifestations of the disease and profuse watery diarrhoea.
The 24-h urine collection for the presence of VMA (metabolites of catecholamines) may be helpful in making the diagnosis of phaeochromocytoma, a rare catecholamine-producing tumour. In adults, 90% of tumours are in the adrenal medulla; 90% of these tumours are benign and 80% are unilateral.
Somatostatin is a peptide that inhibits hormone production by several tumours and its analogue octreotide is useful in the treatment of carcinoid syndrome.
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