Mrs. JP is a 70-year-old woman who was initially diagnosed with ER+ breast cancer 8 years ago. She underwent lumpectomy and hormone therapy but now has advanced to liver metastases. She was given the chemotherapy Epirubicin but developed red urine and injection site soreness so the treatment was stopped. She is now starting Trastuzumab and Paclitaxel immunotherapy and chemotherapy. Trastuzumab targets the HER2 receptor which is overexpressed in her cancer, while Paclitaxel stabilizes microtubules to block cell division.

1. Breast cancer
and Palliative
care
Adam Boulger, Nabeela Alam, Molly
Barden and Joseph Bennington

2. Mrs JP is a 70-year-old active lady who
was initially diagnosed with ER+ breast
cancer 8 years ago. She underwent a
lumpectomy to remove the cancerous
tumor and was prescribed Tamoxifen.
After 5 years, she was taken off Tamoxifen
and prescribed Letrozole for a further 2
years.
Mrs JP Case Update
J

3. Tamoxifen – Estrogen
Receptor Antagonist
Pro-drug Tamoxifen is metabolized mostly by
cytochrome P450’s; CYP26 and CYP3A4 into;
- Non-steroidal triphenylethylene T-metabolites
afimoxifene and endoxifen
- Respective half lives; 14 days & 3 days
T-Metabolites exhibit high selectively towards the
estrogen receptor;
- Antagonistic binding manner to directly inhibit
steroidal estrogen agonist
- Preventing normal estrogen signaling.
A
References: 1,4,5,6,7,11,12,17,18,21,27,28,34,35,37,41

4. Mechanism of Tamoxifen
T-metabolite binding inhibits cancerous ER+ cells from
utilising endogenous estrogen in ER+ oncogene
addiction ceasing estrogen signalling;
- Prevents activation of kinases e.g. Raf, Rac, & P13K
- Involved in multiple pathways such as; NF-KB and
JAK-STAT.
- Prevents anti-apoptotic & proliferative
upregulation
- Prevents aggressive hyperactive immune
environment
- Induces apoptosis and cell shrinkage
Suggested apoptotic effect  3 fold calcium ion
increase;
- Directly damages mitochondria by overloading
- Leads to mitochondrial dysfunction
- Upregulating death signals
M
References: 1,4,5,6,7,11,12,17,18,21,27,28,34,35,37,41

5. Letrozole – Estrogen production inhibitor
Letrozole is a third- generation competitive aromatase inhibitor;
-used in the treatment of ER+ breast cancer.
-blocking aromatase inhibits biosynthesis of estrogen
Highly selective third generation drug;
-Oral, rapid absorbance, 99.9% bioavailability & high potency
-Long half life (t1/2 42hours)
-Elimination by CYP-450 isoenzymes CYP3A4 and CYP2A6 into inactive carbinol metabolite
Potential clash with enzymes here; Letrozole removed via enzymes required
for Tamoxifen metabolism into active molecules..
N
References: 1,4,5,6,7,11,12,17,18,21,27,28,34,35,37,41

6. Mechanism of Letrozole
1. Aromatase catalyzes rate-limiting step in biosynthesis
of estrogen;
- C-19 androgen  C-18 estrogen;
- By utilizing electrons generated by NADPH-cytochrome
P-450 reductase
- Essential residues W224, F221 & M374. Haem essential
for non-covalent binding
2. Letrozole inhibits aromatase by direct competition;
-Preventing androgen binding to substrate-binding site
-Directly inhibits production of C-18 estrogen
-Indirectly represses estrogen signaling
-Prevents activation of kinases which activate; NF-KB &
JAK-STAT for example
Target is Aromatase (cytochrome P-450 enzyme)
A
References: 1,4,5,6,7,11,12,17,18,21,27,28,34,35,37,41

7. Aim of Tamoxifen or Letrozole Treatment
Both monotherapies directly target estrogen signaling by;
-Reduce production of estrogen (Letrozole)
-Reduce binding of estrogen (Tamoxifen)
Both directly reduce proliferation and reduce the risk of the accumulation of malignant mutations.
J
References: 1,4,5,6,7,11,12,17,18,21,27,28,34,35,37,41

8. Tamoxifen vs Letrozole
Both drugs induce the suppression of tumor growth via
alternative mechanisms.
T-metabolites compete with endogenous estrogen for the
estrogen receptors;
- Inhibiting estrogen signalling
Letrozole inhibit the synthesis of estrogen;
- indirectly inhibiting estrogen signalling
Why would the Doctors change Tamoxifen/Letrozole?
Resistance?  Figure
Potentially selecting for aggressive genotypes?
A
References: 1,4,5,6,7,11,12,17,18,21,27,28,34,35,37,41

9. It is discovered Mrs JP now has advanced
breast cancer that has metastasized into
the liver. She is started on a course of
Epirubicin 100 mg/m2 by IV infusion every
3-4 weeks.
Mrs JP Case Update
J

10. What is Epirubicin?
Epirubicin is a 3rd generation anthracycline chemotherapeutic that has
direct effects on DNA;
- Stereoisomer of doxorubicin (2nd gen) with different spatial orientation
of hydroxyl group
- Leads to reduced cardiotoxicity due to reduced free radical production
- Translucent & red solution administered in out-patient facility
- Long half life (t1/2 33 hours)
Epirubicin is a vesicant;
- Causes rapid tissue necrosis & blistering during extravasation.
N
References: 2,3,8,13,24,25

11. Epirubicin;
- forms DNA complex through intercalation of its
planar aromatic rings between nucleotide bases
- cause DNA double or single strand breaks.
- inhibits DNA, RNA and protein synthesis.
Topoisomerase 2b (TOP2B);
- multi-functional enzyme  relieve torsional strain of
the DNA when double strand is separated during
replication.
- nicks the double strand to allow an intact helix to
pass through to relax over-coiled DNA and
subsequently religates the double strand to restore
the DNA structure.
Inhibiting TOP2b prevents this mechanism and the
excess torsional stress breaks the DNA.
Mechanism of Epirubicin
N
References: 2,3,8,13,24,25

12. Alternative Epirubicin mechanisms
Epirubicin induces formation of reactive oxygen species via;
- Series of electron reduction reactions of Epirubicin and its active metabolites
- These cytotoxic free radicals induce cell damage and death
It is also the leading mechanism of epirubicin-derived cardiotoxicity;
- Triggering apoptotic death via complex signalling pathways
J
References: 2,3,8,13,24,25

13. Epirubicin Monitoring
Monitor type Monitor frequency
Full blood count Baseline & before each cycle
Hepatic & Renal function test Baseline, before each cycle & upon clinical indications
Cardiac function test (Echo, RNA and/or MUGA, ECG)
-Monitor for congestive heart failure (CHF)
-Monitor for left ventricular ejection fraction (LVEF)
Baseline & throughout
Clinical toxicity assessment of GI effects, dermatologic
effects, tumour lysis syndrome, infection, bleeding,
ocular effects, local toxicity
Baseline, before each cycle & upon clinical indications
M
References: 2,3,8,13,24,25

14. Epirubicin Cardiotoxicity
Anthracyclines induce dose-related cardiac myocyte injury and
death;
- Leading to left ventricular dysfunction within the heart
- Congestive heart disease
Suggested  by free radical damage due to myocytes being
sensitive to the highly reactive radicals.
Due to epirubicin’s cardiotoxicity, Mrs JP’s heart function must be;
- assessed at baseline
- closely monitored throughout
M
References: 2,3,8,13,24,25

15. Pharmacokinetics of Epirubicin
Administration Direct intravenous: Must be frequently monitored for signs of extravasation. Recommended
dosage is 100-120 mg/m2 every 3-4 weeks. Outpatient
Distribution Rapidly and widely distributed into tissues. Plasma clearance is not affected by the duration of
infusion or administration schedule.
Cross blood brain barrier? No
Volume of distribution 21-27 L/kg
Plasma protein binding 77% (not affected by drug concentrations)
Metabolism Mainly hepatic metabolism; some metabolism occurring in other organs and red blood cells
Active metabolite Epirubicinol- cytotoxic activity is a tenth of epirubicin. Plasma levels lower
than epirubicin
Inactive metabolite Glucuronides of epirubicin and epirubicinol (Glycosidic bonding of
epirubicin or epirubicinol to glucuronic acid)
Excretion Predominantly hepatobiliary; rapid elimination of epirubicin from plasma
Urine 9-10% excreted within 48 hours; 20-27% within 4 days. Red urine is visible 1-2 days
after administration
N
References: 2,3,8,13,24,25

16. Epirubicin plasma protein binding
Epirubicin binds to plasma proteins
to distribute around the body. This
results in a reduction of free,
pharmacologically active
concentrations within the blood.
Plasma clearance in females over
the age of 70 has been shown to
be approximately 35% lower than a
25-year-old female.
This causes a higher proportion of
free active unbound drug present
in the blood during infusion.
Therefore epirubicin must be
closely monitored in elderly
patients due to this as the drug will
have a stronger effect. Lowering
the dosage of epirubicin in elderly
patients has not been investigated.
J
References: 2,3,8,13,24,25

17. Suggestions Why Epirubicin stopped quickly?
1. Allergic or sensitive to Epirubicin
2. Off-target effects  possible extravasation
3. Potentially issues with Mrs JP plasma clearance  age? Hepatic issues? Comorbidities?
4. Potential refusal by patient?
5. Poor performance? Increased progressions  cannot take risks
M
References: 2,3,8,13,24,25

18. After her initial dose of epirubicin Mrs JP reported
passing red urine and claimed the area surrounding
the injection site was very sore and angry looking.
Her doctors decided to discontinue her epirubicin
regimen and start a course of traztuzumab and
paclitaxel.
Mrs JP Case Update
J

19. What is Trastuzumab?
Herceptin: monoclonal antibody immunotherapy highly selective for Her-2 receptor
J
Analytic Test Reasoning
FISH analysis for;
Her-2+ amplification
Her-2 Pathway mutation
Trastuzumab would not be effective if Mrs JP is;
Her-2 negative for amplification
Presents Her-2 pathway mutation
These negate Trastuzumab effect
Echocardiogram for;
Cardiac issues
Trastuzumab can induce cardiac issues.
Not suitable if Mrs JP presents;
- Angina,
- Heart Failure
- Hypertension
What tests are required before administration?
References:9,10, 16,19,20,26,29,31,38,39,40

20. Suggested Mechanisms for Mrs JP’s
Her-2+ amplification acquisition
N
1. Her-2 overexpression is associated by pre-clinical and
clinical resistance to hormonal therapies Tamoxifen and
Letrozole.
2. Epirubicin is mutagenic. Although, Mrs JP received one
cycle.
Case details do not state Mrs JP as Her-2+;
-Trastuzumab treatment is only administered to Her-2+.
Presentation assumes Mrs JP Her-2+
Figure focuses upon Tamoxifen and Letrozole.
A
References:9,10, 16,19,20,26,29,31,38,39,40

21. Why Her-2 Signaling is targeted?
N
Her-2 ligand binding activates and drives;
proliferative signals through a variety of pathways involved
with kinases;
-Ras, Raf, MAPK, P13K and AKT
Her-2+ malignant cells are Her-2+ addicted, preventing
signalling;
- reduces growth
- reduces risk of accumulation of further mutations
Figures do not present dimerization, Her-2 dimerizes with Her-1-4
proteins
References:9,10, 16,19,20,26,29,31,38,39,40

22. Trastuzumab Binding Interaction to
Cellular Target
Trastuzumab binds to domain IV of HER2
C-terminal portion of domain IV consists of three major loops
1st Loop – residues 557-561 – electrostatic interaction
2nd Loop – residues 570-573 – hydrophobic interaction
3rd Loop – residues 593-603 – electrostatic interaction
1st & 2nd loop encoded by exon 17, 3rd encoded by exon 18
A
References:9,10, 16,19,20,26,29,31,38,39,40

23. Trastuzumab Mechanisms of Action
• 1. HER2 degradation
• 2. Antibody-dependent cellular cytotoxicity
• 3. MAPK & PI3K/AKT interference
M
References:9,10, 16,19,20,26,29,31,38,39,40

24. 1. HER2 Degradation
Herceptin binding initiates Her-2 degradation
Herceptin binding non-specific mechanism;
- Clustering of Her-2’s
- Internalization of Her-2
- Early endosome formed
- Lysosomal Degradation
Specific mechanistic process unknown
J
References:9,10, 16,19,20,26,29,31,38,39,40

25. Trastuzumab binding to Her-2;
- Tags Her-2+ cells to immune cells
- Clynes et al  natural killer cells destroy Her-2+ cells by
CD-16 mediated ADCC
Critical associated issues;
- Many cells express Her-2, not just breast cells
- Futuristic approach  produce breast cell specific Her-2+
But - Higher concentration of Her-2+ due to amplification so
higher relative selectivity of Her-2+
2. Antibody-dependent
cellular cytotoxicity (ADCC)
A
References:9,10, 16,19,20,26,29,31,38,39,40

26. 3. MAPK/PI3K/AKT interference
Trastuzumab binding inhibits MAPK & PI3K/AKT
1. Supression of P13k/Akt Pathway by;
- Suppressing Akt phosphorylation by P13K
- Increases PTEN activity/level which negatively
regulates P13k/Akt
Example of action;
• Suppresses Src tyrosine 213 phosphorylation prevents
Src signaling which promotes survival, angiogenesis,
proliferation & metastasis
• Decreases Bcl-XL protein which is anti-apoptotic
2. Supression of MAPK pathway by
• Preventing phosphorylation of MAPK
• Research suggests potentially in relation to RAS/RAC
inhibiting <– mutation key in Trastuzumab resistance
Restores p27 which directly inhibits CDK2 activation.
Directly suppresses cell cycle.
A
References:9,10, 16,19,20,26,29,31,38,39,40

27. Aim of Trastuzumab Treatment
To destroy malignant breast cancer cells with a high selectivity whilst
inducing minimal side-effects by;
- Targeting cells which overexpress HER2
- Reducing Her-2-induced responses
Increasing prognostic outcome for Mrs JP;
- Improving quality of life by reducing symptoms
- Improving progression-free survival
N
References:9,10, 16,19,20,26,29,31,38,39,40

28. What is Paclitaxel-
Mitotic Inhibitor
Paclitaxel is a chemotherapeutic that has direct
effects on the microtubules;
M
References:9,10, 16,19,20,26,29,31,38,39,40

29. Mechanism of Paclitaxel:
Targeting microtubules
Paclitaxel binds to beta-tubulin;
- Reversible binding to drug-binding pocket relies upon
- 3 essential residue-zones for Paclitaxel binding are
- 1-31, 217-233 & Arg282
Beta-tubulin isotype III presented in some malignant cell
lines, Missense mutation at β218A, p.T218A;
- Prevents paclitaxel binding
Paclitaxel binding induces microtubule stabilization;
- Protects from disassembly by inhibiting depolymerisation
- Chromosomes unable to achieve metaphase spindle
configuration
- Blocks mitotic progression
- Triggers apoptosis or induces mitotic arrest by permanent
G0-phase arrest
A
References:9,10, 16,19,20,26,29,31,38,39,40

30. Aim of Paclitaxel Treatment
To destroy malignant breast cancer cells by;
- Targeting cells which proliferate rapidly
- Targeting the expansion of Mrs JP’s malignancy
Increasing prognostic outcome for Mrs JP;
- Improving quality of life by reducing symptoms
N
References:9,10, 16,19,20,26,29,31,38,39,40

31. Trastuzumab:Paclitaxel
combination treatment
M
Why was Mrs JP prescribed combination Trastuzumab-Paclitaxel?
Reduces risk of mutations which inactivates one drug by;
If Trastuzumab resistance acquired, Paclitaxel will initiate killing
mechanisms
No biochemical method to identify whether Trastuzumab therapy
success
Two completely different mechanisms of killing;
- Unlikely one mutation could prevent both
Critical Questions?
Was Mrs JP presenting Beta-tubulin isotype III ?
Was Mrs JP presenting Her-2 pathway mutations for rapid
growth?
Was Mrs JP presenting Her-2 mutation, preventing Trastuzumab
binding?
References:9,10, 16,19,20,26,29,31,38,39,40

32. Critical Issues with Single
Trastuzumab Treatment
A
Mrs. JP’s malignant genotype is not stated;
-High heterogeneity across breast cancer malignancies
- In-vitro analysis identified 1000-fold reduction in interaction between
Her2:Trastuzumab by removing one hydrogen in the interface binding
region.
Khoury et al suggest mutations in the Trastuzumab binding site do not
account for the high frequency of Trastuzumab resistance;
- Specific mutation missense p.H559A could affect binding efficacy?
- However, similar functionality between H&A amino acids
Pathway Mutations prevent Trastuzumab function – e.g. Ras, Raf, P13K;
No current biomarkers for activity. How do we know it is functional?
No clinically approved analysis to identify specific Trastuzumab
resistance before administration
How do we know Mrs JP is not resistant?
- Sequence genomes of those exhibiting Trastuzumab resistances vs
non-resistant?
References:9,10, 16,19,20,26,29,31,38,39,40

33. Over the next few weeks, Mrs JP detoriate rapidly
presenting; 25% reduction in mass, jaundice, swollen
and severely painful abdomen.
Upon liver function tests Mrs JP presents raised;
serum Alanine Transaminase, Aspartate transaminase,
Bilirubin and low serum albumin levels
Mrs JP’s chemotherapy is stopped.
Mrs JP Case Update
J

34. Why did Mrs JP’s
therapy not work?
Suggestions after Tamoxifen/Letrozole;
- Her-2 direct mutation  potential loss of
hydrogen
- Her-2 pathway mutation -> Constitutive activation
- Epirubicin potentially mutagenesis
OR
Is Mrs JP’s cancer just too aggressive?
Are there mutations for alternative growth
signalling?
N
References:9,10, 16,19,20,26,29,31,38,39,40

35. Mrs JP is severely ill and is likely to pass within
the next 12 months.
Palliative care plan has been initiated:
- Twice Daily oral 60mg morphine
- Aim is to improve the quality of life by
easing the symptoms presented
Mrs JP Case Update
J

36. Opioids analgesics act on opioid receptors;
- Majority exhibit high selective affinity towards mu opioid
receptors in the CNS & periphery (over Kappa/delta)
Agonistic binding upon receptors, mimic endogenous opioids;
- Effectively blocking the pain signals from the site of trauma
to the dorsal horn
- Stimulating descending inhibitory modulation of pain
What are Opioid Analgesics?
How will Opioids reduce Mrs JP’s pain?
N
References:14,15,22,23,30,32,33,36

37. Mechanism of Action: Opioid Analgesics
Opioid binds to the neuronal opioid receptor induces;
- α-sub unit exchanges bound GDP with intracellular GTP  Activation
- Dissociation of α-sub unit from βγ complex
- Both α-GTP and βγ can activate target proteins
Closes Ca2+ channels & Opens K+ channels;
- Hyperpolarization of neurone
- Reduces neurotransmitter release
- Reduces cAMP
- Inhibits adenylate cyclase
- Preventing propagation of excitatory signal
M
References:14,15,22,23,30,32,33,36

38. Is Morphine suitable for Mrs JP?
1. Mrs JP presenting reduced serum albumin
potentially could shift the equilibrium of toxic
effects, shifting Mrs JP past TD50 dosages
concentrations;
- Increased bioavailability  increased action
2. Morphine twice 60mg daily via oral;
- Current standard of care for palliative care
3. Morphine metabolised mostly by hepatic
processes;
-Mrs JP’s hepatic issues may increase half life
-Potentially reducing dosage
-Potentially extending dosage intervals
-Patient specific analysis will identify if
required
4. Morphine (& most opioids in general)
induce opioid induced constipation;
-Potential co-administration with specific
laxatives; e.g. Methylnaltrexone antagonist
-Quaternary amine of naltrexone; does not
cross BBB
-Exerts antagonistic effects upon receptors
which opioids bind to in periphery
-Reduces non-target effects
A
References:14,15,22,23,30,32,33,36

39. Alternative Analgesics for Mrs JP
Analgesics Reasoning for Administration
Fentanyl 100 times more potent than morphine – lower dose to achieve therapeutic efficacy
500 times more lipid soluble than morphine – rapid and extensive distribution
Potentially administered via transdermal patch – maintain analgesic action
Reduction in non-target effects such as; nausea
Shorter half-life than morphine
Tramadol Respiratory depression does not occur at same severity as morphine
Reduced occurrence of nausea
Inhibits serotonin reuptake  increases euphoria
Buprenorphine Partial opioid agonist, effective at lower plasma protein concentrations
Transdermal, preventing intravenous
Reduced Mu2 activity
Median survival of 1–14 months for stage IV. Progressive pain plan should be implemented
Potentially paracetamol, depending upon hepatic analysis
M
References:14,15,22,23,30,32,33,36

40. Patient specific discussions will identify
Mrs JP’s personal requirements which the
medical treatment plan should adapt to.
This specific case identifies the demand
for specific genome sequencing to identify
and repress malignancies which acquire
specific mutations which reduce or repress
the action of specific drugs.
Concluding Mrs JP
J

41. Breast cancer
and Palliative
care
Adam Boulger, Nabeela Alam, Molly
Barden and Joseph Bennington