Biological basis of cancer therapy

Question 1

Where is cancer incidence heavily concentrated

Answer 1

In the western world- better detection, unhealthy lifestyle

Question 2

What are the top four most common cancer deaths in males worldwide (2008

Answer 2

Lung
Liver
Stomach
Colorectum

Question 3

What are the top four most common cancer deaths in females worldwide (2008)

Answer 3

Breast
Lung
Colorectum
Cervix

Question 4

Nowadays, why is the morality of breast and cervical cancers less than that of lung cancers

Answer 4

Better diagnosis and screening techniques for breast and cervical cancers.
Lung cancers often diagnosed late and so less patients can be treated surgically.

Question 5

Describe how the incidence of cancers is set to change

Answer 5

22 million cases in 2030
Greater westernisation of developing countries will reduce infection-based cancers (cervical, stomach etc) and increase western cancers such as breast, colorectal, lung and prostate
Better screening and diagnostic techniques- A.I assisted mammography analyser

Question 6

What are the 5 most common cancers worldwide

Answer 6

Lung 
Breast 
Bowel 
Prostate 
Stomach

Question 7

State the main anti-cancer treatment modalities

Answer 7

Radiotherapy
Chemotherapy
Surgery
Immunotherapy

May use radiotherapy/chemotherapy to down-stage the cancer before surgery if it has not yet spread or used when surgery is difficult (i.e too much bleeding, surgeon can’t get well demarcated edges to resect the cancer)

Not many immunotherapies to treat breast cancer.

Question 8

Ultimately, what type of disease is cancer

Answer 8

Cancer is a disease of the genome

Question 9

Describe the main types of genetic mutations that can cause cancer

Answer 9

Chromosome translocation
Gene amplification (copy number variation)
Point mutations within promoter or enhancer regions of genes
Deletions or insertions
Epigenetic alterations to gene expression
Can be inherited

Often when the tumour is sequenced, many genetic mutations are evident.

Question 10

What is important to remember about the genetics of cancer

Answer 10

Cancers are genetically “messy” – so attacking their DNA is a good idea
However, we want to minimise damage to normal DNA in the other normal cells too.

Question 11

How was chemotherapy discovered

Answer 11

1943…John Harvey…100 tons mustard gas bombs…direct hit from German JU88 air raid….explosion..628 suffered from mustard gas…69 died within 2 weeks

Question 12

What are the two different types of systemic therapies to treat cancers

Answer 12

Cytotoxic chemotherapies

Targeted therapies

Question 13

What are the different classes of cytotoxic chemotherapies

Answer 13

1) Alkylating agents
2) Antimetabolites
3) Anthracyclines
4) Vinca alkaloids and taxanes
5) Topoisomerase inhibitors

Often use multiple different classes to minimise anti-cytoxic resistance.

Question 14

What are the different types of targeted therapies

Answer 14

Small molecule inhibitors

Monoclonal antibodies

Question 15

What do the cyto-toxic agents target

Answer 15

Cytotoxics “select” rapidly dividing cells by targeting their structures (mostly the DNA)- except for taxanes and vinca-alkaloids which target microtubules.

Question 16

Describe the administration of cytotoxic chemotherapy

Answer 16

Given intravenously or by mouth (occasionally)
Works systemically
Non “targeted” – affects all rapidly dividing cells in the body
Will therefore lead to hair loss, bone marrow suppression , gut mucosa- can lead to mucositis (mouth ulcers)
Bone marrow suppression causing anaemia, neutropaenia and thrombocytopaenia

Question 17

Describe the uses of cytotoxic chemotherapy

Answer 17

Given post-operatively: adjuvant- to get rid off any cells not removed by the surgery

Pre-operatively: neoadjuvant- chemosensitive cancer cells can shrink tumour before surgery- can lead to more manageable surgery i.e wide local excision instead of mastectomy
As monotherapy or in combination
with curative or palliative intent- but still can have prognosis of 3 years

Question 18

Describe how alkylating agents work

Answer 18

Add alkyl (CNH2N+1) groups to guanine residues in DNA
Cross-link (intra, inter, DNA-protein) DNA strands and prevents DNA from uncoiling at replication
Trigger apoptosis (via checkpoint pathway)
Encourage miss-pairing - oncogenic

Question 19

State some examples of alkylating agents

Answer 19

Alkylating agents: Chlorambucil, cyclophosphamide, dacarbazine, temozolomide.

Question 20

Describe how pseudo-alkylating agents work

Answer 20

Add platinum to guanine residues in DNA
Same mechanism of cell death as akylating agents
Examples: carboplatin, cisplatin, oxaliplatin

Question 21

What are the side effects of alkylating and pseudo-alkylating agents

Answer 21

Side effects: cause hair loss (not carboplatin), nephrotoxicity, neurotoxicity, ototoxicity (platinums), nausea, vomiting, diarrhoea, immunosuppression, tiredness

Oncogenic- so risk of secondary malignancy bur benefit > risk

Question 22

Summarise the mechanism of action of anti-metabolites

Answer 22

Masquerade as purine or pyrimidine residues leading to inhibition of DNA synthesis, DNA double strand breaks and apoptosis

Question 23

Describe the function of anti-metabolites

Answer 23

Block DNA replication (DNA-DNA) and transcription (DNA –RNA)
Can be purine (adenine and guanine), pyrimidine (thymine/uracil and cytosine) or folate antagonists (which inhibit dihydrofolate reductase required to make folic acid, an important building block for all nucleic acids – especially thymine)

Force the DNA into checkpoint — apoptosis

Question 24

Describe some examples of anti-metabolites

Answer 24

Examples include methotrexate (folate), 6-mercaptopurine, decarbazine and fludarabine (purine), 5-fluorouracil, capecitabine, gemcitabine (pyrimidine)

Question 25

Describe the side effects of anti-metabolites

Answer 25

Hair loss (alopecia) – not 5FU or capecitabine Bone marrow suppression causing anaemia, neutropenia and thrombocytopenia Increased risk of neutropenic sepsis (and death) or bleeding Nausea and vomiting (dehydration) Mucositis and diarrhoea Palmar-plantar erythrodysesthesia (PPE) -peeling and reddening of the skin Fatigue

Question 26

Describe the function of anthracyclines

Answer 26

Inhibit transcription and replication by intercalating (i.e. inserting between) nucleotides within the DNA/RNA strand. Also block DNA repair - mutagenic They create DNA and cell membrane damaging free oxygen radicals Examples: doxorubicin, epirubicin Intercalating agent (aromatic)

Question 27

Describe the side effects of anthracyclines

Answer 27

``` Cardiac toxicity (arrythmias, heart failure) – probably due to damage induced by free radicals Alopecia Neutropenia Nausea and Vomiting Fatigue Skin changes Red urine (doxorubicin “the red devil”) ```

Question 28

Describe how vinca alkaloids and taxanes work

Answer 28

Originally derived from natural sources Work by inhibiting assembly (vinca alkaloids) or disassembly (taxanes) of mitotic microtubules causing dividing cells to undergo mitotic arrest Vincas and indibulin- inhibit assembly Taxane (paclitaxel)- inhibit dissasembly

Question 29

Describe the side effects of microtubule targeting drugs

Answer 29

Nerve damage: peripheral neuropathy, autonomic neuropathy Hair loss Nausea Vomiting Bone marrow suppression (neutropenia, anaemia etc) Arthralgia Allergy

Question 30

Describe the physiological role of topoisomerase

Answer 30

Topoisomerases are required to prevent DNA torsional strain during DNA replication and transcription They induce temporary single strand (topo1) or double strand (topo2) breaks in the phosphodiester backbone of DNA They protect the free ends of DNA from aberrant recombination events May induce apoptosis at checkpoints

Question 31

Describe how topoisomerase inhibitors work

Answer 31

Drugs such as anthracyclines have anti-topoisomerase effects through their action on DNA Specific topoisomerase inhibitors include Topotecan and irinotecan (topo I) and etoposide (topo II) alter binding of the complex to DNA and allow permanent DNA breaks

Question 32

Describe the side effects of topoisomerase inhibitors

Answer 32

``` (irinotecan): Acute cholinergic type syndrome – diarrhoea, abdominal cramps and diaphoresis (sweating). Therefore given with atropine Hair loss Nausea, vomiting Fatigue Bone marrow suppression ```

Question 33

What is the key difference between the action of topoisomerase and topoisomerase inhibitors

Answer 33

Topoisomerase is responsible for the unwinding of DNA and they induce temporary single and double strand breaks in the phosphodiester backbone Topoisomerase inhibitors alter the binding of topoisomerase to DNA and allow permanent breaks in the DNA

Question 34

Describe some specific and non-specific topoisomerase inhibitors

Answer 34

 Other drugs such as anthracyclines have anti-topoisomerase effects through their action on DNA.  Specific topoisomerase inhibitor drugs – Topotecan, Irinotecan (topo1), Etoposide (topo2).

Question 35

What will the DNA damage checkpoint and double strand breaks lead to

Answer 35

Apoptosis! | using p53, bcl-2

Question 36

This lady received FEC chemotherapy, 5-fluorouracil, epirubicin and cyclophosphamide for her breast cancer What is the expected benefit and what side effects should she expect?

Answer 36

Treatment will reduce her chance of relapse with the disease by 30% and chance of dying from the disease by 20%. Is it worth the toxicity? We would say yes! We have good therapies to deal with side effects

Question 37

How can we treat the side effects of anti-metabolites such as 5-FU

Answer 37

Pyridoxine (vitamin B6) Antiemetics Transfusions/ platelets / GCSF/ dose reduction Mouth washes Loperamide

Question 38

How can we treat the side effects of anthracyclines such as epirubicin

Answer 38

Irreversible cardiac toxicity but can cap dosing Scalp cooling same as anti-metabolites

Question 39

Outline the timeline of systemic therapy

Answer 39

``` 1940-1950 Nitrogen mustards Alkylating agents Antimetabolites 1950s Vinca Alkaloids 1960s Combination regimens Taxanes and topoisomerase inhibitors Cisplatin 1970s Anthracyclines and topoisomerase inhibitors 1980s Refinement of taxanes ``` 1980s - 1990s Better supportive care: GCSF, bone marrow transplants, anti-emetics Hormone therapies: tamoxifen Imatinib (Glivec)

Question 40

Describe how response rates to cytoxics in ovarian cancer patients has improved

Answer 40

Carboplatin / paclitaxel >80% from 1995 | Up from 45% in 1965 with Chlorambuci

Question 41

Describe how survival retest cytotoxics with ovarian cancer has improved

Answer 41

now over 45 months | originally 20 months

Question 42

Describe some resistance mechanisms to cytotoxics

Answer 42

Drug effluxed from the cell by ATP-binding cassette (ABC) transporters DNA adducts replaced by Base Excision repair (using PARP) DNA repair mechanisms upregulated and DNA damage is repaired -negating the effects of double strand breaks.

Question 43

Summarise how we are manipulating what we know about cancer cells

Answer 43

Modern, targeted (ie non-cytotoxic) therapies seek to manipulate what we know about cancer cells Mainly using monoclonal antibodies and small molecule inhibitors

Question 44

Summarise targeted chemotherapy

Answer 44

 Cancer cells have internal pathways which can be targeted in treatment. o In monogenic cancers, this is fine and treatable. o In others, parallel pathways or feedback cascades become activated- Upreg Ras/Raf and PI3K pathways  “Dual kinase inhibitors” can prevent the feedback loop and this target not just monogenic cancers (picture). Prevent feedback loops but increase toxicities – new therapeutic strategies required - as these pathways are important in all cells

Question 45

What were the original 6 hallmarks of cancer cells

Answer 45

``` Self –sufficient Insensitive to anti-growth signals Anti-apoptotic Pro-invasive and metastatic Pro-angiogenic Non-senescent ```

Question 46

Describe the current 10 hallmarks of cancer

Answer 46

``` Self –sufficient Insensitive to anti-growth signals Anti-apoptotic Pro-invasive and metastatic Pro-angiogenic Non-senescent Dysregulated metabolism Evades the immune system Unstable DNA Inflammation ```

Question 47

Summarise the role of growth singnals

Answer 47

Normal cells need growth signals to move from a quiescent (resting) to active proliferating state These signals are transmitted into the cell via growth factors binding transmembrane receptors and activating downstream signalling pathways Cancer cells are self-sufficient in growth signals- once they become dysregulated- need growth factor initially

Question 48

Summarise the importance of receptor tyrosine kinase in cancers

Answer 48

 Receptor tyrosine kinase = >50% of human malignancies.

Question 49

Give some examples that are over expressed in cancers

Answer 49

HER2 – amplified and over-expressed in 25% breast cancer EGFR – over-expressed in breast and colorectal cancer PDGFR- glioma (brain cancer) Kinase cascade and signal amplification will increase

Question 50

Describe some ligands that are over-expressed in cancers

Answer 50

VEGF – prostate cancer, kidney cancer, breast cancer Kinase cascade and signal amplification will increase

Question 51

Describe constitutive (ligand independent) receptor activation in cancer

Answer 51

EGFR (lung cancer) FGFR (head and neck cancers, myeloma) Kinase cascade and signal amplification

Question 52

Describe the different types of monoclonal antibodies

Answer 52

- momab (derived from mouse antibodies) - ximab (chimeric) e.g cetuximab - zumab (humanised) e.g. bevacizumab trastuzumab - mumab (fully human) e.g. panitumumab

Question 53

Describe the structure of humanised monoclonal antibodies

Answer 53

``` Humanized monoclonal antibody, murine regions (black) interspersed within the light (light gray) and heavy (dark gray) chains of the Fab portion ```

Question 54

Describe the structure of murine monoclonal antibodies

Answer 54

``` Chimeric antibody murine component (black) of the variable region of the Fab section is maintained integrally. ```

Question 55

What part of the receptor do monoclonal antibodies target

Answer 55

Monoclonal Antibodies target the extracellular component of the receptor This can: Neutralise the ligand Prevent receptor dimerisation Cause internalisation of receptor

Question 56

What else can mAbs target

Answer 56

mAbs also activate Fcγ-receptor-dependent phagocytosis or cytolysis induces complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC).

Question 57

Describe some examples of monoclonal antibodies used in cancer therapy

Answer 57

Bevacizumab binds and neutralises VEGF. Improves survival in colorectal cancer Cetuximab targets EGFR

Question 58

How do small molecule inhibitors work

Answer 58

Bind to the kinase domain of the tyrosine kinase within the cytoplasm and block autophosphorylation and downstream signalling

Question 59

Describe Glivec (Imatinib) as the first targeted therapy

Answer 59

In 1973 the (9,22) chromosome translocation in patients with CML was discovered (Janet Rowley) Found to create its own unique fusion protein called Bcr-abl, an enzyme which drove over-production of white cells 1996 Buchdunger et al published data on a drug that could specifically target bcr-abl (and not affect other proteins) Fantastic clinical results (90% complete response rates in patients with CML) heralded the departure from “conventional cytotoxics” into a new era of “targeted therapies” Example of “Oncogene Addiction” – uniquely hyperactive oncogene driving a tumour (Achilles’ Heel)

Question 60

Describe how Glivec works

Answer 60

Glivec is a small molecule inhibitor and targets the ATP binding region within the kinase domain Ligand in ATP-binding site, inhibiting kinase activity of ABL1 Not all cancers are monogenic - i.e tyrosine kinase domain of ABL1

Question 61

Where can the small molecule inhibitors act

Answer 61

Small molecule inhibitors act on receptor TKs but also intracellular kinases – therefore can affect cell signalling pathways

Question 62

Describe the small molecule inhibitors which inhibit receptors

Answer 62

Examples of SMIs inhibiting receptors include erlotinib (EGFR), gefitinib (EGFR), lapatinib (EGFR/HER2), sorafinib (VEGFR)

Question 63

Describe the SMIs which inhibit intracellular kinases

Answer 63

SMIs inhibiting intracellular kinases include: Sorafinib (Raf kinase) Dasatinib (Src kinase) Torcinibs (mTOR inhibitors)

Question 64

Describe the benefits of targeted therapies over cytotoxic

Answer 64

By acting on receptors (either externally or internally), targeted therapies block cancer hallmarks (e.g VEGF inhibitors alter blood flow to a tumour, AKT inhibitors block apoptosis resistance mechanisms) WITHOUT the toxicity observed with cytotoxics

Question 65

What are the advantages of mAbs

Answer 65

``` High target specificity Cause ADCC, complement mediated cytotoxicity and apoptosis induction Can be radiolabelled Cause target receptor internalisation Long half-life (lower dosing frequency) Good for haematological malignancies Liked by regualatory authorities (FDA) ```

Question 66

What are the disadvantages of mAbs

Answer 66

Large and complex structure (low tumour or BBB penetration), less useful against bulky tumours Only useful against targets with extracellular domains Not useful for constitutively activated receptors Cause immunogenicity, allergy Parenteral (IV) administration Risky! (though humanisation reduces risk) Expensive

Question 67

What are the advantages of small molecule inhibitors

Answer 67

Can target TKs without an extracellular domain or which are constitutively activated (ligand independent) Pleiotropic targets (useful in heterogenic tumours/ cross talk) Oral administration Good tissue penetration Cheap

Question 68

What are the disadvantages of small molecule inhibitors

Answer 68

``` Shorter half-life, more frequent administration Pleiotropic targets (more unexpected toxicity) ```

Question 69

What is a key issue with all of the targeted therapies

Answer 69

Resistance ``` Mutations in ATP-binding domain (e.g BCR-Abl fusion gene and ALK gene, targeted by Glivec and crizotinib respectively) Intrinsic resistance (herceptin effective in 85% HER2+ breast cancers, suggesting other driving pathways) Intragenic mutations Upregulation of downstream or parallel pathways ```

Question 70

Describe anti-sense oligonucleotides

Answer 70

Single stranded, chemically modified DNA-like molecule 17-22 nucleotides in length Complementary nucleic acid hybridisation to target gene hindering translation of specific mRNA Recruits RNase H to cleave target mRNA Good for “undruggable” targets Very expensive Can precent translation of ICAM-1

Question 71

Describe the use of RNAi

Answer 71

Single stranded complementary RNA Has lagged behind anti-sense technology –especially in cancer therapy Compounds have to be packaged to prevent degradation - nanotherapeutics CALAA-01 targeted to M2 subunit of ribonucleotide reductase. Phase I clinical trials in cancer –results awaited Again, very expensive

Question 72

Describe the regulation and funding of anti-cancer therapies

Answer 72

Licenced by European Medicines Agency Approved by National Institute for Health & Care Excellence (NICE) Cancer Drugs Fund - £340m/yr until March 2016 Regional differences in accès to drugs

Question 73

What is a major obstacle to the targeted approach

Answer 73

Tumour heterogeneity

Question 74

Describe actionable mutations and driver mutations

Answer 74

Drivers of heterogeneity- identify the driver events for genomic instability that may occur at the nexus of the trunk and branch may provide new approaches to limit tumour diversity and adaptation Actionable mutations - early drivers of disease biology lead to ubiquitous somatic events present in every tumour sub clone and tumour region. such ubiquitous tumour mutations may present more robust therapeutic targets and optimal synthetic lethal targets

Question 75

Describe how we can track heterogeneity and bottlenecks

Answer 75

development of noninvasive techniques to monitor and track the sub clonal dynamics of tumour architecture through treatment may enhance understanding of resistance mechanisms as branches are pruned at the expense of outgrowth of other branches harbouring heterogeneous resistance mutations

Question 76

Describe tumour sampling bias

Answer 76

Biopsies in 1 region of a heterogenous primary or metastatic tumour will identify trunk events but may also identify as many or more heterogeneous events not shared by all regions of the tumour or by all tumour subtypes. Comparison of paired primary/metastatic samples may enhance identification of trunk events for therapeutic targeting Regional genetic ITH may have an impact on ex vivo assays of cell phenotypic function

Question 77

Describe the success story of targeting B-raf

Answer 77

``` Activating mutations of B-Raf identified in 60% melanomas Substitution of glutamic acid for valine (V600E) causes a 500-fold increase in activity B-Raf inhibitor (vemurafenib) showed dramatic Phase I activity in melanoma (80% PR or CR) Extends life span of mutation holders by 7 months (Sosman et al, NEJM, 2012) Side effects arthralgia, skin rash and photosensitivity ```

Question 78

Describe immune modulation via programmed cells eath 1 PD-1

Answer 78

Present on the surface of cancer cells Required to maintain T cell activation After binding the ligand PDL1, the body’s T cells can no longer recognise tumour cells as foreign If either is blocked (ligand or receptor) , immune system is stimulated Nivolumab (developed by BMS) is anti-PD1 antibody

Question 79

What can nivolumab be used to treat

Answer 79

In treatment-refractory melanoma, non-small cell lung cancer (NSCLC), and renal cell carcinoma (RCC) Saw overall response rates of 31% in melanoma (cf the usual 5-15%) Median survival of 16 months (phase I trial)

Question 80

What is important to remember about cancer therapy

Answer 80

We now know that cancer is fundamentally a genetic disease The mainstay of cancer treatment is cytotoxic chemotherapy, but this is toxic, non-specific and sometimes ineffective Glivec was the first rationally-designed agent with impressive results in CML suggesting targeted drugs would replace cytotoxics Targeted agents are generally monoclonal antibodies and small molecule inhibitors Resistance develops to these drugs too, they can show toxicity and promiscuity Can biopsies be believed? Targeted therapies and “personalised oncology” are very expensive!

Question 81

What is meant by a bucket trial

Answer 81

Therapy against mutation not location of cancer

Question 82

Summarise personalised medicine

Answer 82

A 2012 Harris poll of 2760 US patients and physicians found that doctors had recommended personal genetic tests for only 4 per cent of patients (New Scientist, 2013) In July 2013, the UK government announced that it would offer private companies a subsidy from a £300 million fund to encourage investment in its personalised medicine initiative 23andMe (banned by FDA, now approved by MHRA), DeCODEme (Iceland) and Knome (Ozzy Osbourne)

Question 83

Describe the impact of sequencing tumours prior to therapy

Answer 83

Depends on reliable methods – currently not being done (risk of false negative results) Used to provide treatment as well as prognostic information Concentrate on particular pathways for certain cancers? Circulating biomarkers, tumour cells or DNA

Question 84

Describe some new therapeutic avenues

Answer 84

Nanotherapies – delivering cytotoxics more effectively Virtual screening technologies to identify “undruggable” targets Immunotherapies using antigen presenting cells to present “artificial antigens” Targeting cancer metabolism

Question 85

Why do many cancer treatments cause side effects and how can we overcome these

Answer 85

Cytotoxic chemotherapy: cannot select only cancer cells so affects all rapidly dividing cells Common side effects of all chemotherapy: and how to minimise Hair loss - scalp cooling Bone marrow suppression causing anaemia and neutropenia - transfusions/platelets/dose reduction and GCSF Nausea and vomiting - antiemetics Tiredness - cannot be avoided

Question 86

Explain the rational for new cancer drug development t

Answer 86

Tumour heterogeneity: major obstacle to targeted approach Resistance mechanisms for cytotoxics: Enhanced DNA repair Drug efflux from cell using ATP-binding cassette (ABC) transporters DNA adducts replaces by base excision repair using PARP Resistance mechanisms for targeted therapy: Mutations in ATP binding domains Intrinsic resistance Intragenic mutations Upregulation of downstream pathways

Question 87

What are the most common cancers worldwide

Answer 87

 The most common cancers worldwide – lung, breast, bowel, prostate and stomach. o Most common by gender – Lung in men, breast in women. o “Western cancers” include – breast, colorectal, lung and prostate

Question 88

State the different mutations that can lead to cancer

Answer 88

o Chromosome translocation. o Gene amplification (from copy number variations). o Point mutations – in promotor/enhancer regions. o Deletions/insertions. o Epigenetic alterations. o Heritable mutations.