Cellular / Biochemical / Molecular Targets in Breast Cancer

Introduction
Breast cancer is a heterogenous group of disease with wide ranging biological behaviour.
Move toward targeted therapy and personalized medicine is set to accelerate

Molecular Profiling

Intrinsic molecular subtypes of breast cancer
Now clear that breast cancer can be usefully classified by gene expression profiles.
- luminal A, luminal B, HER2, basal

ER +ve vs ER -ve tumour profiling is most robust distinction.
- luminal tumours show expression of ER, lmw cytokeratin 8/18
- luminal A = low grade, high ER expression and excellent prognosis
- luminal B = higher grade, higher proliferation and poorer prognosis

HER2 stands for Human Epidermal growth factor Receptor 2
- usually ER negative, and characterized by over-expression of HER2 and associated genes.
- tend to be more aggressive, however responsive to targeted therapies.
- p53 mutations
- a few (10-15%) are also ER positive and fall into / overlap into luminal B group

Basal group has higher expression of genes characteristic of normal basal cells
- including hmw cytokeratins, HER1 and SM actin.
- generally high-grade, poorly differentiated tumours with a poor prognosis.
- predominantly negative for ER, PR and HER2; this 'triple negative' is a surrogate for basal like classification.
- in reality triple-negative is also heterogenous, 70% basal
- Tend to occur in younger women, in BRCA pts, and in those of African descent 

Amsterdam signature
A set of 70-gene expression profiles that can classify breast cancer prognosis.
- more predictive than nodal status
- commercially available (MammaPrint) and undergoing prospective validation trials.

Oncotype DX
A 21-gene assay using reverse-transcription PCR on RNA isolated from paraffin-embedded tumour specs
Validated in a large multi-center trial
- stratifies patients in terms of prognosis
- appears to guide benefit of adding chemo to tamoxifen in high-risk patients
- potential to guide targeted clinical therapies in at-risk patients

Targeted Molecular Therapies
TARGET
DRUG
STATUS
ER
Tamoxifen
Survival benefit, adjuvant therapy and metastatic disease control.

Aromatase inhibitors
Anastrolozle (arimidex), Letrozone
Survival benefit, adjuvant / metastatic disease. postmenopausal women

Fulvestrant
Second-line therapy; metastatic disease in postmenopausal women
HER2
Trastuzumab (Herceptin)
Survival benefit, adjuvant and metastatic disease
HER1,HER2
Lapatinib
Second-line; metastatic disease; in trials
Also new agents in trials for HER1, VEGF, PART1/2
LHRH blockers - suppresses ovarian fx.

ER Pathways

1. Now recognized that there are 2 ERs: alpha and beta
- alpha is the classic ER, detected on usual assays; 75% are +ve
- beta role is uncertain at this time; high ratios may be assoc. with tamoxifen resistance.
--> activation of nuclear ER receptors is a critical step in the growth of many breast cancers.
--> ER signaling = estradiol binds to ERs in cell and initiates transcription, --> cell proliferation, angiogenesis and metastasis.
Tamoxifen
Selective ER modulator (SERM), competitively binds to ER, blocking estradiol from initiating downstream signaling.
Aromatase inhibitors
Inhibit aromatase, which converts androstenedione to estrodiole peripherally.

2. PR is an estrogen-responsive gene that results from ER activation.
- 55% of ER+ve tumours are also PR+ve

3. Endocrine therapies target ER pathways in several ways
- selective (tamoxifen) interact directly with the ER, binding to inhibit estrogen-dependent transcription while maintaining agonist activity at some sites.
- cf pure antagonists (fulvestrant), binding to ER and degrades the ER protein without agonist activity.
- aromatase inhibitors and oophorectomy do not interact directly with ER but reduce amount of estrogen available to bind as a ligand.

4. ER targeting is remarkably successful.
- adjuvant therapy with 5y of tamoxifen reduces recurrence by 40%-50%
- lowers odds of breast cancer death by 30%;
- similar benefit seen with aromatase inhibitors in postmenopausal females.

Other Targets

1. EGFR
- transmembrane TK receptors, including erbB2 (HER2/neu)

2. HER2 over-expression
- HER2/neu is a gene involved in regulation of cell proliferation
- HER2 protein is transmembrane tyrosine kinase receptor that participates in the growth, differentiation and survival of breast cancer cells.
- is a protoncogene; ie a normal gene with potential to be an oncogene with molecular alterations (mutaiton, amplification or over-expression of its product).
- HER2 protein is over-expressed in about 25% of invasive breast cancers. 
- HER2 amplification is associated with more aggressive phenotype and predicts for response to trastazumab therapy (recombinant monoclonal antibody specific to HER2)
- diagnosed by immunihistochemistry and confirmed by FISH when positive.

3. Trastazumab (herceptin)
- a humanized monoclonal antibody that binds to the extracellular domain of the HER2 protein, inhibiting downstream signalling.
- 5 RCTs from >10000 women randomized showed significant improvement recurrence, metastasis, disease free (50%) and overall (33%) survival
- clearly limited to HER2 +ve tumours
- mechanisms uncertain and many patients subsequently become less responsive.
--> minor cardiotoxicity rate; not a major concern.
- ongoing trials in this space looking at novel inhibitors of HER2
--> Lapatinib current under investigation.

4. PI3K/AKT pathway
- HER family partners, e.g. HER3 necessary for HER2 function; hence are another target
- focus of acquired resistance to HER2
- Activating mutations of HER3 pathways are common; agents under development.

5. EGFR and MAPK
- HER2/EGFR heterodimer is a master regulator controlling carcinoma proliferation.
- targetted with new results in trials.

6. Angiogenesis and Lymphadema targeting
- e.g. VEGF pathways; can be dealt to by a monoclonal antibody
- promising results in early trials.
- highly expressed in inflammatory breast cancer

7. DNA repair pathways
- relevant to BRCA1 and 2
- unable to repair DNA; genomic instability.
- PARP1/2 are enzymes that act as DNA damage sensors
- PARP upregulated in triple negative tumours
- inhibitors of PARP allow theoretically higher efficacy of DNA damaging cancer drugs e.g. cisplatin
- promising results in early trials.

8. Other directions
- toposiomerases; DNA repair
- microRNAs, protein encoding RNSa that are involved in proliferation regulation and a good target
- DNA methylation / epigenetics; associated with some cancer subtypes and explored as a biomarker / target

Endocrine Therapeutic Agents in Practice

1. Breast cancers are highly heterogenous.
- higher concentration of ERs, more likely that anti-estrogen will be beneficial.
- 'any detectable' ER still termed an endocrine-responsive tumour.

2. Tamoxifen remains the cornerstone, but in post-menopausal women, assoc. with
- increased risk of VTE
- increased uterine cancer.

3. Aromatase inhibitors avoid these problems, more effective and less toxic in post-menopausal women
- but(!) paradoxically increase estrogen in premenopausal women.

4. The definition of menopause is:
- prior bilateral oophorectomy
- age 60 or older
- age <60 and amennorhoea for 12+ months
- FSH and estradiol levels in the postmenopausal range

Pre-Menopausal Women

1. 20% of breast cancers.
- 60% ER positive.

2. Tamoxifen well proven in multiple trials and quality meta-analysis evidence
- reduces annual recurrence by 40%   
- and mortality by 34%
- irrespective of age, menopausal status or node status.

3. Suppression of ovarian function? (LHRH)
- currently unclear if there is additional benefit in this;
- trials need updating in context of modern hormonal and adjuvant therapy.
--> guidelines recommend tamoxifen; if contraindicated then consider suppression.

4. Aromatase Inhibitors (AIs) are contraindicated.

Post-Menopausal Women

1. Several large RCTs support AIs
- current guidelines support use of AI as standard hormonal therapy.

2. If AIs contraindicated
--> 5y of tamoxifen.

Targeted Therapy in Practice

1. Trastazumab guidelines say:
- use in all patients with HER2 +ve disease with tumours >1cm
- regardless of horomone receptors

2. If tumours are <1cm, clinical judgement required as no clear evidence in this space.