Unit 6 - Targeted Therapy

Question 1

What is personalised medicine?

Answer 1

An approach to give a specific form of treatment (either drug choice or dose regime) to a patient sub-group in order to maximise treatment efficacy and minimize any side-effects

Question 2

What factors could be responsible for the difference in patient response (HER2+ breast cancer) to neratinib?

Answer 2

• Extrinsic factors (diet, stress – as cortisol can activate cAMP pathway modifying drug response)
• Host genetics – pharmacogenetics – perhaps SNPs in CYP enzymes for instance
• Other tumour characteristics

Question 3

What ways can a molecular driver of tumour growth be identified?

Answer 3

Techniques like NGS or gene microarray – from biopsies or CTC/CFDNA.
Importantly this must be causative not correlative.

Question 4

What are the two types of biomarkers?

Answer 4

Prognostic – indication of patient outcome (e.g., ER+ breast cancer has favourable outcome)
Predictive – suggests a patient might respond to a certain type of treatment/therapy – HER2+ breast cancer for Tratuzumab or olaparib with BRCA mutated ovarian cancer

Question 5

Describe the ways we can analyse biomarkers

Answer 5

IHC for ER/PR
NGS for specific mutations like L858R in NSCLC
Oncotype DX looks at 21 genes associated with different processes in breast cancer – (compared to reference) and then determines with there is a high risk of recurrence and subsequently the severity of treatment (chemotherapy vs anti-hormones)

Question 6

What are the differences between nonsynonymous and synonymous mutation and how could this be important in drug response?

Answer 6

A non-synonymous mutation changes the residue in the protein, whilst a synonymous mutation does not!
This could either effect protein structure and activity or potentially affect promoter activity or mRNA conformation/stability

Question 7

Give examples of where SNPs in non-target genes can result in changes in drug activity

Answer 7

• DPYD and 5-FU – Dihydropyrimidine dehydrogenase which detoxifies 5-FU – common T>G reduces its activity resulting in increased activity and toxicity
• CYP2D6 and tamoxifen – enzyme helps metabolise tamoxifen to endoxifen (active form) – hence mutations may reduce activity
• XRCC1 and platinum agents – increased activity will mean cells are more able to repair DNA damage – hence will change sensitivity
• ABC1/MDR1 – alters ability for cell to efflux drug – therefore will impact on cellular concentration and activity
  Note the increased activity of the drug may increase efficacy and/or toxicity and decrease will obviously decrease its efficacy.

Question 8

Give examples of how somatic mutations in tumours may effect drug response

Answer 8

GISTs often have KIT mutations (MAPK/PI3K…) which can be inhibited by imatinib – those with exon 11 mutations respond better to those with exon 9 or no mutation!

Question 9

Discuss the problems that exist with personalised medicine approaches

Answer 9

• Tumours are very heterogenous so only certain populations may respond resulting in tumour evolution and resistance
• The biopsy quality of tumours is questionable as around 20% cannot be used for biomarker assessment!

Question 10

What types of NSCLC are most common and which of these are used by smoking?

Answer 10

Non-squamous are 70% - non-smoking whist Squamous are 30% - caused by smoking

Question 11

Discuss how the stage of NSCLC effects what treatment is chosen

Answer 11

Stage 0 – removal by surgery
Stage I-IIIA – surgery and adjuvant chemotherapy or chemotherapy and radiotherapy
Stage IIIB – chemotherapy and radiotherapy
Stage IV – SACT – usually involving platinum doublet (cisplatin + pemetrexed)

Question 12

Outline the 3 ways of targeted therapy in NSCLC.

Answer 12

• A target associated with a particular phenotype – EGFR inhibitors in east Asian female, non-smokers, or adenocarcinoma patients (much better response rate)
• Target a process central for cancer progression – bevacizumab in angiogenesis – alongside immunotherapy and platinum doublet or nintedanib
• A particular molecular sub-group based on mutation profiling – certain mutations within EGFR

Question 13

What were the 1st line EGFR inhibitors?

Answer 13

Gefitinib and Erlotinib (competitive reversible inhibitors) 😉 form an acneiform rash - THESE ARE ANILOQUINAZOLINES

Question 14

Why is bevacizumab given alongside immunotherapy?

Answer 14

VEGF is an immunomodulator which decreases T-cell activity, hence immunotherapy is used to recover immune activity

Question 15

List a mutation of EGFR that are associated with a)drug sensitivity, b) primary drug resistance and c)mutations associated with acquired resistance

Answer 15

a) Exon 19 deletion, L858R – 100x more sensitive to 1st generation than wild-type tumours (70% response rate in those VS very low in WT)
b) Exon 20 insertions
c) T790M

30% of NSCLCs have activating EGFRs

Question 16

How common are EGFR mutations in NSCLC and where do they most commonly occur?

Answer 16

Around 30% of patients – with mutations most commonly in exons 19-21 – TK domain (mostly exon 19 deletion of L858R)

Question 17

What is the impact of the L858R mutation?

Answer 17

This residue is within the activation loop – which regulates the binding of ATP to the active site – this charge change results in a conformational change which promotes the open/active form – increasing the enzymes activity – but also making it far more sensitive to these first generation inhibitors compared to the wild-type molecule!!
X100 times for sensitive to the 1st generation TKI’s

Question 18

What are the 2nd generation EGFR inhibitors?

Answer 18

Afatinib (blocks all EGFR family) and dacomitinib – have improved PFS compared to 1st generation – also competitive inhibitors - but irreversible because they form covalent bonds

Question 19

How often does resistance to the 1st generation EGFR inhibitors occur and describe the impact of one of these secondary mutations

Answer 19

Most patients see resistance within 12 months and about 50% have the T790M mutations. This is the gate keeper residue – increased size sterically blocks the entry of the inhibitors but not ATP into the active site.
This also increases its affinity for ATP – increasing oncogenic signalling!!

Question 20

Describe some of the ways EGFR inhibitor resistance occurs (1st generation)

Answer 20

• Parallel RTK hyperactivation via mutations/amplification of ligand or receptor – HER2, MET (can also interact with HER3) -crizotinib
• Downstream pathway activation
  a. Kras (12/13/61) – in younger, western or smokers with adenocarcinoma
  b. BRAF – in 2% - dabrafenib
  c. PI3KCA/PTEN mutations

Question 21

Why are 2nd generation EGFR inhibitors less affected by T790M compared to 1st generation?

Answer 21

This is because they can still form covalent interactions with C797 which increases its affinity – but still unsuccessful at tolerable doses

Question 22

Name a 3rd generation EGFR inhibitor

Answer 22

Osimertinib – binds irreversibly and inhibits the T790M mutants and is less effective against the WT (hence less cytotoxic) – forms a covalent bond with C797 -Can cross the BBB

Question 23

Describe a way we can monitor patients for EGFR secondary mutations?

Answer 23

Using liquid biopsies with cfDNA

Question 24

What mutations lead to resistance against Osimertinib?

Answer 24

C797S – prevents this covalent bond that is required for its activity!!

Question 25

What other mutations are seen in lung cancer and how can they be targeted (apart from EGFR)?

Answer 25

Met – crizotinib
KRAS – smokers – AMG510
BRAF – smokers- dabrafenib
PI3K
ALK – younger, men, non-smokers — gene fusions with EML4 – active receptor tyrosine kinase via dimerization enhancement->MAPK and PI3K – crizotinib (L1196M – resistance), 2nd generation alectinib (resistance via G1202R), 3rd generation loralatinib (can across BBB)
ROS-1 – RTK - fusion gene ->PI3K/MAPK -can use crizotinib (G202R4/gatekeeper resistance) or Entrectinib.

Question 26

What treatments are available for NSCLC patients who have no mutations which are associated with targeted therapy?

Answer 26

75% of patients Immune checkpoint inhibitors like pembrolizumab/nivolumab (anti-PD-1 – prevents interaction with PD-L1) – prevent T-cell exhaustion/inactivation. Atezolizumab is anti-PDL1 – and can potentially work in those regardless of PD-L1
Can be used in mutation positive with TKIs/bevacizumab/platinum chemotherapy

Question 27

Why is treatment of NSCLC so difficult?

Answer 27

• Most patients present with late stages of the disease – already very advanced
• There are lots of pathways which can be switched between for resistance
• Brain metastases is very common – and many molecules don’t cross the BBB

Question 28

What are the risk factors for HCC?

Answer 28

• Underlying chronic liver disease/cirrhosis – like haemochromatosis – cirrhosis – regeneration and inflammation which can promote tumorigenesis
• Obesity, drug use, anabolic steroids, drinking and smoking
• Viruses – Hepatitis B/C

Question 29

How do hepatitis B and C cause cancer?

Answer 29

Hepatitis B
Responsible for around 80% of cases – increases risk-100-fold. DSDNA virus encoding a weakly oncogenic protein X
Hepatitis C
Transfected via blood – increases risk 20-fold – Positive strand- RNA virus can cause cancer in many ways:
- X protein can induce DNA transcription alterations – BCL-2 and miRNAs (that regulate HIF-1-alpha/MMPs?) promoting proliferation
- Can induce DNA damage like p53 (G->T mutation) as work in synergy with aflatoxin B1
- Viruses induce chronic inflammation – GFs like PDGF or IL’s and can also promote angiogenesis (infecting endothelial cells)
-

Question 30

How can HCC be prevented?

Answer 30

• Hepatitis B vaccination
• Infection control (screening transfusions)
• Anti-viral treatments
• Screening at risk cohorts
• Screening patients with chronic HCV/HBC
• Lifestyle changes – drinking/smoking/drug use

Question 31

How is liver cancer staged?

Answer 31

Using the Barcelona clinic liver classification system
condition.
- Stage 0 – single nodule and ≤ 2cm
- Early stage (A)- single node or ≤ 3 nodules ≤ 3cm and ECOG0
- Intermediate stage (B)– Multinodular but ECOG 0
- Advanced Stage – Portal Invasion N1, M1, ECOG 1-2
- Terminal Stage (D)
Can also look at increase alpha-fetoprotein in the blood

Question 32

How is stage 1 HCC treated?

Answer 32

This stage is operable which means they have good survival rates. There are 3 types of surgery:
Operable disease – completely removed the liver with a transplant if possible (confined disease)
Partial resection – removes entire cancerous lesion (liver can re-generate)
Ex-vivo liver resection – removal of disease area and reimplantation of remaining tumour-free liver

Question 33

How is stage B HCC treated?

Answer 33

Transarterial chemoembolization – TACE – where chemotherapy (doxorubicin/cisplatin) is injected via a catheter into the hepatic artery to cut off blood flow causing ischemia and a high level of drug in the liver (minimises toxicity)
Bland/Transarterial embolization – TAE – particles injected via catheter to block hepatic blood flow
Transarterial chemoembolization with drug-eluting beads – DEB-TACE – beads block blood flow and elute chemotherapy
Necrosis is never complete, can rely on portal blood supply and angiogenesis can promote tumour survival.

Question 34

Describe the many ways in which stage C HCC is treated

Answer 34

HCC associated with many RTK hyperactivations as well as angiogenesis – because of this many pan-RTK inhibitors are used.
- Sorafenib – RAF1, BRAF, VEGFR2, PDGFRβ – MAIN THERAPY
- Lenvatinib – VEGFR1-3, PDGFRα, FGFR1-4 and RET
- Regorafenib – VEGFR1-3, BRAF V600, PDGFR, FGFR (after progression on sorafenib – attacks the pathways that may lead to resistance
- Cabozanitinib – MET, VEGFRs and KIT – targets MET+
Resistance is common in these therapies as they can switch signalling to other RTKs and they are associated with severe toxicities like hypertension, diarrhoea or hand-foot syndrome
Ramucirumab – VEGFR2 antibody
Atezolizumab – PDL1 antibody with bevacizumab
Nivolumab – PD1 inhibitor

Question 35

What are the reasons why brain tumours are difficult to treat?

Answer 35

• Tumours are localised in the brain so difficult to remove surgically
• Limited capacity of the brain to repair itself
• Disrupted blood supply can reduce drug delivery and may lead to accumulation of fluid around the tumour – intracranial hypertension
• Therapeutic insensitivity
• Treatment neurotoxicity
• They are able to activate many different pathways leading to resistance as well as significant tumour heterogeneity!

Question 36

What cells do gliomas originate from?

Answer 36

Astrocytes, oligodendrocytes, Schwann cells]]] glial cells - non-neuronal cells that maintain homeostasis (neurotransmitter/ions), maintain the blood-brain barrier and produce myelin

Question 37

Discuss the mechanism of temozolomide and what is a predictive factor for its efficacy?

Answer 37

Temozolomide is an DNA damaging agent which can help add a methyl-group to guanosine bases at position O6- this can lead to a substitution as it forms a base-pair with T.
The methylation status of methylguanine DNA methyltransferase is key to this as this enzyme carries out the removal of this methyl group – hence promoter methylation and gene silencing will lead to a better response to treatment.
Used in brain cancer

Question 38

What mutations are common in glioblastomas?

Answer 38

• RTK (EGFR/PDGFR), Ras, PI3K in 88% (as well as PTEN loss)
• P53 altered in 87% (or CDKN2A inactivation- inhibitor of CDK INHIBITOR 2A that regulates P16 and P14 which are TS, inhibiting CDK4/6 and activating p53 respectively)
• Rb inactivation in 77%

Question 39

What targeted therapies exist for glioblastoma treatment?

Answer 39

Targeting EGFR – common EGFRVIII mutation/loss of exons 2-7 (ligand-binding domain) or activate in autocrine manner
1st generation gefitinib and erlotinib not associated with good outcome due to cross talk with IGFR and poor intratumoural levels/pharmacology
Antibodies like cetuximab are too large to cross the BBB
2nd generation TKIs under investigation like lapatinib
PDGFR – itself and ligand overexpressed
Imatinib show poor efficacy as well as antibody ramucirumab
RAS
Farnesyltransferase inhibitors like lonafarnib as well as MEK inhibitors like sorafenib
PI3K/Akt
Perfosine – poor toxicity

Question 40

Describe the approaches of targeted therapy in melanoma

Answer 40

BRAF inhibition – mutations in around 60%
BRAF is a main component of MAPK signalling – the majority of cases are associated with the V600E mutations ->MAPK pathway but also NF-κB and A/C-RAFs
Inhibitors like vemurafenib
Resistance occurs via RTK hyperactivation to regulate other pathways are mutations in MEK/RAS to hyperactivate the MAPK pathway
Immunotherapy
The anti-CTLA4 (0n the T-cells) ipilimumab to prevent interaction with CD80/86 (B7 receptor) involved in T-cell inactivation
Same for Pembrolizumab (anti-PD1) and nivolumab (anti-PD1) – T-cells

Question 41

Describe the activation of the ERK pathway

Answer 41

• RTK-ligand binding leads to receptor dimerization, phosphorylation and activation
• GRB2 binding helps bind the GEF SOS which can activate Ras (via transformation from inactive GDP-from to GTP-form)
• This can then phosphorylate and activate RAF->MEK->ERK -> expression of cyclin D1

Question 42

Explain why the combination of immunotherapy and targeted therapy in melanoma might be a good approach

Answer 42

Because immunotherapies see improvements later on in treatment regimes, whilst targeted therapies are preferential short-term

Question 43

Describe the two types of ovarian cancer and which type of cell is usually responsible.

Answer 43

Type I – chemoresistant but low grade – KRAS/PI3K/ARD1A (part of SWI/SNF of chromatin remodelling complex)
Type II – chemo sensitive and usually associated with BRCA/P53 mutations lots of different mutations in small amounts - meaning its difficult to target all (IGFR/RAS/Notch)
Both associated with epithelial cells

Question 44

Describe the type of approaches of targeted therapy in ovarian cancer.

Answer 44

Angiogenesis – inhibitors like cediranib and bevacizumab
PARP inhibitors in those with BRCA mutations
MEK inhibitors like selumetinib due to mutations in BRAF/KRAS and PTEN or BRAF/VEGFR inhibitor sorafenib

Question 45

Describe the concept of synthetic lethality

Answer 45

Were the loss of function of two genes (usually involved in a similar process) together, but not individually will lead to cell death

Question 46

Describe how PARP inhibitors work in those with BRCA mutations

Answer 46

BRCA1//2 play a role in double-strand repair by helping recruit molecules like RAD51 to help repair the DNA using the second copy of the chromosome in a process known as homologous recombination. Tumour cells that have mutations in these genes are less able to repair DNA as they instead use an error prone process – NHEJ which can often lead to cell death
PARP is an enzyme that helps repair single-strand breaks via BER – recruiting molecules like XRCC1 to the damage site. The use of competitive PARP inhibitors like olaparib will prevent single strand breaks from being repaired in ALL cells. Normal cells have functional HR pathway so will be able to repair the subsequent double-strand breaks so will be largely unaffected – however tumour cells will have to use NHEJ – which will lead to tumour cell death
Resistance is quite common though through restoration of HR or Increased PARP activity
Is a possibility can be used in patients with mutations in other HR genes like PALB2 or RAD51 which cumulatively constitute a significant proportion of patient

Question 47

Explain the ways in which ovarian cancer cells can become resistant to chemotherapies

Answer 47

BRCA1/2 can affect sensitivity to platinum based agents as they help repair any DNA damage the agent produces
Signalling pathways like PI3K up-regulated (Either via RTK or intracellular kinase)
Efflux proteins like p-glycoprotein

Question 48

Describe the possible treatments for TNBC?

Answer 48

Immunotherapies like pembrolizumab or atezolizumab
PARP inhibitors like olaparib in those with BRCA mutations
ADCs – like Sacituzumab govitecan – Trop2 (expressed by lots of TNBC) and a topoisomerase inhibitor
PI3K inhibition like ipatasertib or Capivasertib

Question 49

231. Describe the two functions of the pancreas and what two cell types are involved

Answer 49

Exocrine – regulate digestion
Acinar cells secrete chymotyprsinogen, lipases and amylases
Ductal cells secrete bicarbonate to regulate the pH
Endocrine – regulates glucose metabolism
α/β cell secrete glucagon and insulin
PP and Δ cells regulator secretory activity of other cells through pancreatic polypeptide which inhibits enzyme secretion and somatostatin effects neurotransmitters

Question 50

What are the risk factors associated with pancreatic cancer

Answer 50

• Age
• Smoking/alcohol/poor diet
• Chronic pancreatitis
• H.pylori infection
• Genes like APC/BRCA/P53

Question 51

In pancreatic cancer what is PANIN?

Answer 51

Pancreatic intraepithelial neoplasia – ERBB/KRAS….INK4….P53 mutations….Telomerase

Question 52

What types of non-targeted therapy are used in pancreatic cancer

Answer 52

Whipple procedure – not many can have this can procedure is complex
Chemotherapy like carboplatin, 5-FU and docetaxel
Radiotherapy – brachytherapy

Question 53

What kinds of targeted therapy exist in pancreatic cancer?

Answer 53

MEK inhibitor pimasertib to inhibit those with RAS mutations which are an early event and found in many pancreatic cancer patients
Anti-IGFR antibody dalotuzumab – hyperactivation is also common and autocrine/paracrine activity
SMO inhibitors like saridegib

Question 54

Explain how the hedgehog pathway can promote pancreatic cancer progression

Answer 54

Patch receptor inhibits SMO which will lead to the cos-2 complex binding to Gli, cleaving it so it cannot enter the nucleus
SHH binding to patch relinquishes this inhibition meaning the Cos2 complex is bound to Smo. Gli can then enter the nucleus and transcribe genes like mTOR, EGF and TGFβ.
SHH can be overexpressed in cancer due to Ras hyperactivity and TAFs

Question 55

Describe the risks associated with CRC?

Answer 55

80% non-inherited 
-	Age
-	Type II diabetes 
-	Obesity 
-	Smoking/alcohol 
-	High fat/red meat
20% hereditary 
-	FAP – APC mutations 
-	Lynch syndrome – Mut genes MMR

Question 56

What is the most common early event in CRC development?#

Answer 56

Loss of APC – promotes beta-catenin signalling.

Can be followed both other changes like loss of p53 and activation of KRAS/RAF/PI3K as well as epigenetic changes

Question 57

What are the four stages of Duke staging in CRC?

Answer 57

• A – confined to mucosa
• B -Perforates outer serosa
• C – lymph nodes close to bowel
• D – distant metastasis

Question 58

Describe the non-targeted chemotherapeutic approaches in CRC

Answer 58

• The first agent will likely be capecitabine (anti-metabolite ->5-FU) +/- oxaliplatin (XELOX) – DNA strand breaks, or 5-FU (thymidyl synthase inhibitor) + folonic acid (enhance 5-FU toxicity as stabilises enzyme complex) +/- oxaliplatin (FOLFOX)
• 2nd line therapy is 5-FU + folonic acid + irinotecan (FOLFIRI) topoisomerase inhibitor or irinotecan monotherapy if FOLFOX 1ST line

Question 59

What are the targeted therapy agents used in CRC?

Answer 59

EGFR is overexpressed in 80% of patients
Cetuximab and panitumumab – prevent ligand binding and dimerization (G1 arrest) – have shown potential with FOLFOX and FOLFIRI
Anti-angiogenic agents
Bevacizumab – binds to VEGF-A- again can be used alongside FOLFIRI – resistance occurs via KRAS/BRAF mutations or via chronic treatment resulting in an angiogenic switch
Aflibercept is an anti-VEGF recombinant protein attached to an FC-region
Immune checkpoint inhibitors
MMR proteins (MSH2/6 mutation or MLH1 promoter methylation) can lead to mutations resulting in abnormal protein production that trigger T-cell infiltration
Pembrolizumab and nivolumab (PD-1 antibodies) can activate T-CELLs as many of these tumour cells will also produce PDL1 to cause T-cell exhaustion

Question 60

Describe the mechanisms CRC patients have to become resistance to EGFR therapies.

Answer 60

Non-genetic:
- Increased EGFR//EGF expression
- Altered binding affinity of EGFR ligands or extracellular domain mutations
- Dysregulation of EGFR internalisation
- Overexpression of other receptors like HER2/MET
- PI3K/AKT up-regulation
Genomic
- BRAF mutations – V600E – co-treat with Encorafenib – really important to determine appropriate therapy as not-responsive to EGFR treatment
- RAS mutations like G12C – is REALLY IMPORTANT to determine if chemo/EGFR therapy used!!!! Mutants do not respond to EGFR therapy - hence sequencing is commonly used to detect

Question 61

What is Lonsurf?

Answer 61

The use of the nucleoside analogue trifluridine and the thymidine phosphorylase inhibitor tipiracil -given after failure of EGFR inhibitors in CRC