CBIO 9: Cancer Resistance, Biomarkers and Personalised Treatment

Question 1

Observe the learning outcomes of this session

Answer 1

Question 2

What is cancer therapeutic resistance?

Answer 2

• Cancer therapeutic resistance occurs when cancers develop resistance to treatments such as chemotherapy, radiotherapy and targeted therapies, through various mechanisms.
• These may include specific genetic and epigenetic changes in the cancer cell(s) and/or the microenvironment* in which the cancer cells reside.

Question 3

Define tumour microenvironment

Answer 3

• the tumour microenvironment includes non-cancerous cells within and adjacent to the tumour, and the proteins expressed by them (including extracellular matrix), that may contribute to tumour growth.
• For example, increased matrix stiffness of hepatocellular carcinoma cells promotes resistance to chemotherapy.

Question 4

How can we overcome therapeutic resistance in cancer?

Answer 4

• Using an alternative drug that works via different mechanisms. This is probably the most obvious approach.
• Understanding exactly how drug resistance develops in cancer.
  
  • This allows for the possibility of designing drugs to specifically target it.
• Use a combination of therapies to target different aspects of the tumour.
  
  • This reduces the opportunities for the tumour to become resistant.
• By personalising treatment.
  
  • Choosing a treatment that is tailored to a specific patient significantly reduces the rate of therapeutic resistance.
• We came across examples of therapy resistance, and targeted therapies,

Question 5

Is drug resistance a problem for traditional chemotherapy?

Answer 5

• The prognosis for cancer patients who suffer from metastatic cancer, and who depend on systemic drug therapy (like chemotherapy), remains poor despite many advances in cancer therapy.
• Drug resistance is a serious problem for traditional chemotherapy; 90% of cases of chemotherapy failure are related to drug resistance.
• Resistance to targeted therapy is also a major concern.

Question 6

What are the different types of drug resistance?

Answer 6

• Drug resistance can be intrinsic or acquired.
• Intrinsic means the drug resistance mechanism exists prior to treatment, acquired means drug resistance is induced (selected for) by drug treatment.

Question 7

What is pharmacodynamics?

Answer 7

• how a drug affects the organism

Question 8

What is pharmacokinetics?

Answer 8

• how the organism affects the drug

Question 9

Describe the general principles of drug resistance

• pharmacokinetic (PK) factors
• pharmacodynamic (PD) factors

Answer 9

• Pharmacokinetic (PK) factors such as drug absorption, distribution, metabolism and elimination limit the amount of a systemically administered drug that reaches the tumour.
• Pharmacodynamic (PD) factors are features cancer cells have which limit the anticancer activity of the drug.

Question 10

How could the efficacy of a drug be limited?

Answer 10

• due to poor drug influx or excessive efflux
• drug inactivation or lack of activation
• expression level changes of the drug target
• activation of adaptive pro-survival responses
• no induction of cell death due to dysfunctional apoptosis

Question 11

Use this diagram to summarise the main mechanisms of drug resistance

Answer 11

• decreased drug uptake
• increased drug efflux
• aberrant metabolism
• alterations to drug target
• enhanced DNA damage repair
• apoptosis suppression
• collateral sensitivity

Question 12

Explain the mechanism of drug resistance: decreased drug uptake

Answer 12

• Drug resistance can be due to decreased uptake/influx, which means cancer cells prevent the drug from entering them.
• Examples are:
• Mutation of one or both of the folate transporters: Folate is a vitamin that enters cells using the folate transporters found on cell membranes.
  
  • The anti-cancer drug, methotrexate (a toxic folate analogue) uses folate receptors to enter cancer cells and mutations in these transporters leads to drug resistance.
• Mutation of nucleoside transporters: Nucleoside analogues (anti-cancer drugs that are similar to nucleotides
  
  • e.g. gemcitabine and 5-fluorouracil) use specific transporters.
  • Mutations in these will lead to drug resistance.
• Reduced expression of receptors and transporters on cell membranes by decreased endocytosis
  
  • e.g. Copper transporter 1 (CTR1). CTR1 transports cisplatin into the cells and reduced levels lead to cisplatin-resistance in cell line models.

Question 13

Explain the mechanism of drug resistance: increased drug efflux

Answer 13

• drug resistance can be due to increased drug efflux, which means cancer cells remove drugs that have entered the cell.
• Example:
• P-glycoprotein (Pgp), also known as MDR1 (multi-drug resistance protein 1), is part of the ATP-binding cassette (ABC) transporter family of proteins
• As the name suggests, it is an ATP binding protein pump located in the cell membrane that pumps many foreign substances out of cells.
• Over-expression of MDR1 is associated with drug resistance to Pgp substrates e.g. the chemotherapy drugs Doxorubicin, taxanes and vinca alkaloids.
• Drug inhibitors of Pgp, such as verapamil, can reverse drug resistance in vitro.
• The clinical significance of MDR1 over-expression is however disputed.
• There is good evidence to suggest that over-expression of this protein plays a significant role in drug resistance in cases of acute myeloid leukaemia (AML) and myeloma, but its role in drug resistance where solid tumours are concerned remains unclear.

Question 14

Explain the mechanism of drug resistance: aberrant metabolism

Answer 14

• Drug resistance can be due to altered drug metabolism to increase detoxification, which means the cancer drugs are no longer toxic. Examples are:
• Glutathione (GSH) metabolism: GST maintains cellular redox homeostasis by being a is a powerful anti-oxidant that protects the cells against the damaging effects of reactive oxygen species.
• ROS production is significantly increased in cancer cells because of mitochondrial dysfunction, altered metabolism, and frequent genetic mutations.
• Therefore, as an adaptive response, cancer cells harbour elevated levels of ROS-scavenging molecules such as GSH.
• Elevated levels of GSH in tumour cells are able to protect cancer cells by conferring resistance to several chemotherapeutic drugs.
• GSH conjugates to platinum chemotherapy drugs (e.g. oxaliplatin and cisplatin) and modifies them to substrates for ABC transporters, which facilitate drug efflux.
• CYP450, an enzyme found in the liver, can inactivate irinotecan (Topoisomerase I inhibitor, used to treat colon cancer).
• Metallothionein (MT), a protein found on the membrane of Golgi apparatus, also binds platinum drugs and inactivates them.
• Drug resistance can be due to altered drug metabolism to decrease activation, which means cancer drugs are not activated within cancer cells. An example is:
• Cytarabine (also known as AraC), is a nucleoside analogue widely used for the treatment of acute myeloid leukaemia (AML) which requires phosphorylation by the enzyme deoxycytidine kinase to be activated.
• Resistance to cytarabine develops when levels of deoxycytidine kinase are reduced, through downregulation of the protein or a mutation in the gene which limits it’s function.

Question 15

Explain the mechanism of drug resistance: alterations to drug target

Answer 15

• The efficacy of a drug is influenced by its molecular target.
• Alterations of the drug target, such as mutations or altered expression levels, can promote development of drug resistance.

For example:

• Gleevec (imatinib) is a tyrosine kinase inhibitor that targets the fusion oncogene, BCR/ABL protein in chronic myeloid leukaemia.
• Drug resistance develops as a result of mutations occurring at the binding site of the drug within the BCR/ABL protein.

Question 16

Explain the mechanism of drug resistance: enhanced DNA damage repair

Answer 16

• drug resistance can be due to changes in DNA repair pathways. For example:
• Resistance to the DNA damage-inducing drug cisplatin occurs due to enhancement in DNA repair mechanisms.
• This can be due to high expression of important components such as the protein, Excision repair cross-complementing group 1 (ERCC1), an important factor in the NER (nucleotide excision repair) DNA repair pathway.
• ERCC1 has been implicated as a predictor of resistance to cisplatin in ovarian cancer.

Question 17

Explain the mechanism of drug resistance: apoptosis suppression

Answer 17

• Drug resistance can be due to changes that result in suppression of apoptotic pathways. Examples are:
• inactivating mutations in genes coding for apoptotic proteins, such as p53.
• activating mutations in genes coding for anti-apoptotic proteins, such as Bcl-2.

Question 18

Explain the mechanism of drug resistance: collateral sensitivity

Answer 18

• Collateral sensitivity occurs when resistance to one drug confers hypersensitivity to an alternate cytotoxic agent, to which parental cells were not originally sensitive.
• The same genetic alteration that caused resistance to one drug now sensitises them to another.
• One example is given below (see also the concept of synthetic lethality in CBIO8):
• A patient with metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer was resistant to crizotinib because of a mutation in the ALK kinase domain.
• The patient responded to another drug, lorlatinib. When the tumour relapsed, sequencing of the tumour revealed an ALK mutation which conferred resistance to lorlatinib in addition to the mutation conferring resistance to crizotinib.
• However, the new mutation enhanced binding to crizotinib, negating the effect of first mutation and re-sensitising resistant cancer to crizotinib.
• The patient received crizotinib again, and cancer-related symptoms resolved.

Question 19

What are cancer stem cells?

Answer 19

• Although their existence remains slightly controversial, cancer stem cells (CSCs, or cancer cells with stem-cell-like properties) are (or are postulated to be) rare immortal cells within a tumour that can both self-renew by dividing
• and also give rise to many cell types that constitute the tumour and can therefore form tumours.
• Only a small subset of cells can give rise to a new tumour.

Question 20

Why are cancer stem cells an important target population for anticancer therapeutics?

Answer 20

CSCs represent an important target population for anticancer therapeutics as their survival following therapy is highly likely to result in disease relapse.

Question 21

Why are CSCs believed to be highly resistant to conventional chemotherapies?

Answer 21

• High expression of ATP-binding cassette (ABC) transporter proteins
• High aldehyde dehydrogenase (ALDH) activity, which oxidises and detoxifies several substrates
• Expression of anti-apoptotic proteins such as Bcl-2 and Bcl-XL
• Enhanced DNA damage repair
• Activation of key pro-survival signalling molecules such as NOTCH and nuclear factor-κB (NF-κB)
• Relatively quiescent – chemotherapy targets rapidly dividing cells

Question 22

What are the models of drug resistance mechanisms

Answer 22

a) Somatic mutation model - due to somatic mutation, a tumour may contain a mixture of drug sensitive and drug insensitive cells.
- Following chemotherapy, the drug insensitive cells will continue to grow (causing relapse) and remain resistant to any further chemotherapy.
b) Cancer stem cells – since cytotoxic agents (chemotherapy) primarily affective proliferating cells, they will not affect cancer stem cells that are in a quiescent state even in rapidly proliferating tumours.
- These cells therefore show a degree of drug insensitivity relative to cycling cells and might persist at the end of chemotherapy.
- Subsequent relapse is then due to the re-growth of persistent stem cells that were predominantly in G0.
c) In practice, chemoresistance develops despite initial chemosensitivity and it therefore seems likely that both models presented in (a) and (b) underlie much of clinical drug resistance.
- These two processes presumably occur concurrently, but the relative proportions vary from individual to individual, and determine the clinical pattern of relapse and drug sensitivity.

Question 23

What does a high rate of epigenetic change in cancer cells lead to?

Answer 23

• There is often a high rate of epigenetic change occurring in cancer cells, which creates diversity in gene expression and, during drug treatment, could lead to the development of acquired drug resistance.

Question 24

What are epigenetically poised persistant tumour sustaining cells?

Answer 24

• When a tumour containing cells with diverse gene expression (known as heterogeneity) is treated with a drug, most cells will die.
• However some cells may become resistant due to epigenetic changes and will survive, and eventually this subpopulation will expand.
• This leads to tolerance of treatment
• these are epigenetically poised persistant tumour-sustaining cells
• because chromatin domains have both the activation-associated histone modification and the repression-associated modification.
• Therefore, if the chromatin modification that confers tolerance to drug is lost, the tolerance can be reversed.

Question 25

What are the two possible outcomes for epigenetically poised persistent tumour sustaining cells?

Answer 25

1. If tolerance is reversed, tumour cells will become sensitive to the drug again (chemo-sensitive relapse).
2. However, exposure of poised cells to further chemotherapy and selection can ‘lock in’ an epigenetic state and fix gene expression.
   - This results in proliferation of a drug-resistant population of tumour cells (chemo-resistant relapse).

Question 26

Many anticancer drugs require metabolic activation and thus cancer cells can develop resistance through decreased drug activation.

True or False?

Answer 26

• true

Question 27

What is the meaning of ‘altered drug targets’ in drug resistance?

Answer 27

• Changes, such as mutations or modifications of expression levels, of the drug target that lead to cancer cell death

Question 28

What are epigenetically poised persistent tumour sustaining cells?

Answer 28

• Cells in which chromatin domains have both the activation-associated histone modification and the repression-associated modification

Question 29

What are cancer biomarkers?

Answer 29

• Cancer biomarkers are substances produced by cancer cells, or by other cells of the body in response to cancer.
• Most tumour markers are made by normal cells as well as by cancer cells; however, they are may be produced at much higher (or sometimes lower) levels under cancerous conditions.

Question 30

What are the characteristics of cancer biomarkers?

Answer 30

• Some biomarkers can be detected in the circulation (whole blood, serum, or plasma), or in excretions or secretions (stool, urine, sputum, ejaculate, or nipple discharge).
• In these cases, the presence of the biomarker can be assessed and/or or changes in its levels measured easily over time.
• Due to the way the samples are taken collection is relatively non-invasive.
• However, other biomarkers are within tissue, and require either a biopsy or special imaging so that they can be evaluated.
• Cancer biomarkers are biomolecules such as DNA, RNA, proteins, peptides, and biomolecule chemical modifications.
• The alterations associated with cancer can be due to a number of factors, including germline or somatic mutations, transcriptional and epigenetic changes, and post-translational modifications.
• A collection (termed a signature), of transcriptomic (gene expression), proteomic, or metabolic changes can also serve as biomarkers.
• Biomarkers typically differentiate between an affected patient from a person without the disease and/or they may also distinguish between different stages of disease.
• Many different tumour markers have been characterised and are in clinical use.
• Some are associated with only one type of cancer, whereas others are associated with two or more cancer types.
• No “universal” tumour marker which can detect any type of cancer has been found.

Question 31

What are the limitations of tumour biomarkers?

Answer 31

• Sometimes, non-cancerous conditions can cause the levels of certain tumour biomarkers to increase, meaning the biomarker is not specific for cancer.
• An example is prostate specific antigen (PSA) for prostate cancer, which you learnt about in CBIO6 where levels of PSA can be raised in benign prostatic hyperplasia.
• Furthermore, not everyone with a particular type of cancer will have a higher level of a particular tumour marker associated with that cancer.
• Moreover, tumour markers have not been identified for every type of cancer.

Question 32

What are the potential applications in cancer treatment?

Answer 32

1. Estimating risk of developing cancer
2. Screening for signs of primary cancer
3. Diagnostic
4. Prognostic
5. Predicting whether a therapy will work for a given patient
6. Monitoring

Question 33

Describe how biomarkers can estimate risk of developing cancer

Answer 33

• Biomarkers have been identified that can be used to determine an individual’s risk of developing cancer.
• For example, a woman with a strong family history of ovarian cancer can undergo genetic testing to determine if she is a carrier of a predisposing germline mutation, such as loss-of-function mutation of BRCA1, which will increase her risk of developing breast and/or ovarian cancer (Easton et al, 1995, Am J Hum Genet 56: 265–271).
• If so, she can opt for more intensive screening, chemoprevention with tamoxifen (a type of hormone therapy), or prophylactic (preventative) surgery.
• Remember a germline mutation in BRCA1 increases risk but does not mean for sure the pa will develop the disease (why? think back to earlier modules).

Question 34

Describe how biomarkers can screen for cancer

Answer 34

• Biomarkers can be used to screen otherwise healthy patients for malignancy.
• Screening programmes are often offered to older people who are more at risk of developing cancer.
• An example is the use of the gFOB (guaiac Fecal Occult Blood) test in bowel cancer screening for those over 55 years old.
• The gFOB test detects hidden blood (fecal occult blood) in faeces.
• This could also be termed early diagnostic testing.

Question 35

Describe how biomarkers can be used to diagnose cancer

Answer 35

• A diagnostic biomarker is used to identify whether a patient has cancer.
• For example, if a patient is found to have a lung nodule on their chest CT scan, histological evaluation of the biopsy specimen can determine whether the tissue is cancerous.
• Another example is the discovery of the BCR-ABL fusion gene (Philadelphia chromosome) in blood or bone marrow, which is used to confirm a diagnosis of chronic myeloid leukaemia.

Question 36

Describe how biomarkers can be used for cancer prognosis

Answer 36

• “Prognosis” means the likely outcome of the disease.
• In patients who have been diagnosed with a cancer, biomarkers can help determine prognosis (likely outcome), disease progression or likelihood of disease recurrence.
• Traditionally, the clinicopathologic characteristics (signs and symptoms) of a tumour were used to determine the prognosis.
• More recently, newer technologies are being utilised to assess prognosis for individual tumours.
• For example, in breast cancer, there are a number of gene expression signatures developed that can be used to estimate prognosis for an individual patient based on analysing the tissue from tumour.
• The best characterised of which is called the 21-gene recurrence score.

Question 37

Describe how biomarkers can be used to predict treatment outcome

Answer 37

• Biomarkers can also be used to predict how well a patient will respond to a given treatment, or for determining which therapy is likely to be most effective.
• In colorectal cancer, KRAS is a predictive biomarker, because somatic mutations in KRAS are associated with poor response to anti-epidermal growth factor receptor (EGFR) directed therapies.

Question 38

Describe how biomarkers can be used to monitor cancer

Answer 38

• Biomarkers can be used to monitor status of the disease, either to detect recurrence or determine response or progression to therapy.
• For example, Carcinoembryonic antigen (CEA) is monitored in colorectal cancer and some other cancers to keep track of how well cancer treatments are working or check if cancer has come back.

Question 39

Have a look at the table of biomarkers currently being used in a range of cancer types

Answer 39

Question 40

How do you discover biomarkers?

Answer 40

• Gene expression profiling - the measurement of the expression of thousands of genes at once which creates a global picture of cellular function.
• This can be done by analysing tumour tissues from patient cohorts using RNA-sequencing.
• MS-based profiling – using mass spectrometry (MS) to profile (often small) metabolites in a tissue (for an example see the iKnife later on).
• Peptidomics - using mass spectrometry, this is the direct measurement of the endogenous peptides present in a given biological sample.
• Biomarker family – this approach is sometimes employed if a member of a protein family is already an established biomarker and scientists will study other members of that family as they may also be good cancer biomarkers.
• Secreted factors – proteins or nucleic acids which are secreted by cancer cells that can be found in the surrounding environment of tumour or in blood.
• Protein arrays - this is a method used to monitor the levels and activities of proteins on a large scale.
• Auto-antibodies – tumour-associated antigens could serve as biosensors for cancer because tumours naturally elicit an immune response in the host and produce auto-antibodies.
• MS-imaging of tissue - this is a technique used to visualise the spatial distribution of molecules by their molecular masses.
• Gene fusion/translocations – studying whether there have been gene fusion due to a chromosomal translocation (in all cancers not just haematological cancers).
• Serum proteomics – using mass spectrometry to identify the protein composition of serum (also applicable to other biofluids).

Question 41

What are the 5 conceptual phases in cancer biomarker discovery and development?

Answer 41

1. Preclinical exploratory studies
   - In this phase, tumour and non-tumour specimens are compared to find differences between them.
   - If strong evidence is found this will generate hypotheses for clinical tests to detect cancer.
   - Strategies for cancer biomarker discovery discussed above can be used in this phase.
2. Assay development and validation
   - A clinical assay that uses a specimen of choice (preferably something that can be obtained non-invasively, or with minimal invasion) is developed in this phase.
   - The assay must discriminate individuals with cancer from those without.
   - The patients assessed in this phase have established disease.
   - The utility of the assay in detecting early disease is not demonstrated in this phase.
3. Retrospective longitudinal clinical repository studies
   - Specimens are collected and stored from a cohort of healthy (at the time) individuals who were monitored for development of cancer.
   - Using these specimens is its demonstrated that the biomarker has the capacity to detect preclinical disease and criteria for ‘positive’ screening results are defined.
4. Prospective screening studies
   - In this phase, individuals are screened with the assay together with the use of established diagnostic procedures to those who screened positive.
   - (This can help to establish the tumour stage or the nature of the disease at the time of detection).
5. Randomised control trials
   - A non-biased study in which a number of similar people are randomly assigned to a group where they are screened for the biomarker or assigned to a group where they are not screened.
   - The objective of this phase is to determine if screening reduces the burden of cancer in the population

Question 42

Describe the iKnife

Answer 42

The iKnife burns tissue as it cuts, and the gaseous molecules are collected and identified in real time by a mass spectrometer (see diagram and video below).

The molecular pattern will distinguish whether the tissue is normal or malignant. If it is malignant the surgeon can remove it immediately, without the need for secondary surgery. This avoids the problem of residual tumour left behind after surgery.

In the case of the iKnife, the biomarkers used to distinguish between normal and malignant tissue are phospholipids that compose cellular membranes. An important hallmark of cancer is uncontrolled cellular proliferation, requiring an increased rate of biological membrane synthesis that is orders-of-magnitude higher than healthy tissue. Consequently, phospholipid biosynthesis pathways differ between healthy and malignant tissues.

Question 43

What is personalised treatment?

Answer 43

• personalised treatment, also known as stratified medicine, classifies tumours according to their genetic makeup instead of where they grow in the body
• this information can be used to strategise a treatment plan for a particular patient

Question 44

What is the holy trinity of personalised cancer therapy?

Answer 44

• Identifying who to treat:
• this is achieved by identifying the exact mechanism of how a given treatment kills tumour cells and using this information to develop biomarkers to identify those patients in which the treatment will work
• Combating drug resistance:
• achieved by understanding the mechanisms behind treatment resistance and developing biomarkers to predict when this will occur
• as well as using clinical approaches to prevent or delay resistance
• Optimising combination therapy:
• achieved by understanding why some drug combinations work, how combinations can be used to combat drug resitance and finding predictive markers to show if the combinations are working in patients

Question 45

Describe adoptive cell transfer (ACT) a form of personalised immunotherapy

Answer 45

• In essence, tumour cells from this patient were DNA sequenced to identify mutations.
• T-cells were isolated from the same patient’s blood and exposed to these tumour mutations via APC cells.
• Following this, the T-cells were cultured with a small piece of the patient’s tumour to activate them.
• The activated T-cells were selected, expanded and reinfused back into the patient.
• The activated T-cells specific for the tumour mutation will seek, infiltrate and mount an immune attack against tumour cells in this patient.