1. Chemotherapeutic Treatment Options for Triple Negative Breast Cancer
Lauren Barney
April 17, 2013

2. Breast Cancer Subtypes
Breast cancer is classified into clinical subtypes based upon receptor expression
These subtypes dictate possible therapeutic options and vary in their prognosis
Luminal: derived from the luminal cells
ER+, PR+
Can use hormonal therapy
Less aggressive
Basal: derived from myoepithelial cells
ER-, PR-
No specific target for therapies
More aggressive
HER2-enriched
Luminal A
Luminal B
Claudin-Low
HER2-enriched
Basal
ER: estrogen receptor
PR: progesterone receptor
HER2: human epidermal growth factor receptor 2

3. Luminal and Basal Characteristics
Basement membrane
Myoepithelial Cells  Basal
Luminal Cells  Luminal
Basal
Luminal
Low ER
Low HER2
High CK5/6
c-KIT higher
High EGFR
High p53 mutation
High p53 protein
High cyclin E
Very high vimentin
High ER
Higher HER2
Low CK5/6
Low c-KIT
Low EGFR
Low p53 mutation
Low p53 protein
Low cyclin E
Low vimentin
Cyclin E: Cyclin E binds to G1 phase Cdk2, which is required for the transition from G1 to S phase of the cell cycle that determines cell division
Vimentin: type III intermediate filament protein that is expressed in mesenchymal cells. EMT marker

4. Triple Negative Breast Cancer
15-25% of all breast cancer, but much higher proportion of all breast cancer mortality
Lack ER, PR and HER2 – no targeted therapies
Much more aggressive
Younger age at diagnosis, high grade, large tumor size, aggressive relapse
High proliferation, poor differentiation, basal marker (cytokeratin 5/6) expression, and aggressive clinical course, with early relapse and decreased survival
TN tumors have specific morphologic characteristics: elevated mitotic count, tumor necrosis, pushing margin of invasion, and stromal lymphocytic response and high nuclear-cytoplasmic ratio
Not all basal are TN

5. TN vs Basal Subtypes
The terms triple negative and basal are often used interchangeably in breast cancer subtyping.
Triple negative denotes the lack of ER, PR and HER2 receptors (clinical observation)
Basal describes the tumors that overexpress those genes that characterized breast basal epithelial cells based on gene expression
These often overlap!
Basal-like breast cancer is characterized by certain features that include the TN phenotype, but TN and basal-like are not synonymous terms. A discordance of up to 30% has been described between the two groups.

6. Treatments can be targeted for cancers that express hormonal receptors or HER2; TN remains a clinical challenge.
Hormonal therapy: blocks estrogen activity
Tamoxifen, ER antagonist
Competitively binds to ER & inhibits estrogen effects
HER2 targeted therapy
Herceptin & others
These targeted therapies work really well!
There is no specific target on TN cells! Must use cytotoxic chemotherapeutics, surgery, radiation.
Tamoxifen

7. Current Options for TNBC
Standard course of treatment is very aggressive: surgery with adjuvant and neoadjuvant chemotherapy and radiation therapy
Neoadjuvant: administration of a drug before a main treatment – increases rate of breast conserving therapies and helps to understand a patient’s response to drugs
Adjuvant: any therapy given after primary therapy – used when there is a high risk of recurrence
The search is on for specific targets!

8. TNBC Treatment
Chemotherapy typically includes combinations of taxanes (T), anthracyclines (A), and oxazophorines (C)
Taxanes: disrupt microtubules & inhibit cell division
Paclitaxel, docetaxel
Anthracyclines: most effective chemotherapeutics!
Three mechanisms: inhibit DNA and RNA synthesis, blocks transcription and replication, creates oxygen free radicals
Daunorubicin, doxorubicin, epirubicin, idarubicin
Oxazophorine: DNA alkylating agent
Cyclophosphamide (C)

9. Taxane and Anthracycline Based Therapy
Typical regimens:
AC-T: doxorubicin plus cyclophosphamide every 2 weeks for four cycles followed by docetaxel every 2 weeks for 4 cycles
Investigating taxol before AC (not standard therapy)
TAC: docetaxel, doxorubicin, and cyclophosphamide every 3 weeks for 6 cycles
Different dosing regimens, frequencies can help to improve efficacy
Dense dosing is better (more frequent doses are better)

10. CMF therapy may actually reduce recurrence of TNBC compared to anthracycline or taxane-based treatment
CMF is a much older therapeutic regimen than TAC or AC-T
Cyclophosphamide (alkylating agent, oxazophorine)
Methotrexate (antimetabolite, stops cell growth & division)
Fluorouracil (called 5FU; pyrimidine analog, antimetabolite)
Many different dosing schedules possible

11. TN Tumors are Chemosensitive
Recently, studies have shown that TNBC is more responsive to anthracycline or anthracycline/taxane chemotherapy than Luminal subtypes
Patients who had a complete response to chemotherapy had good prognosis regardless of subtype
Despite this, TNBC patients still have a worse distant disease free-survival and a poor prognosis
Result of high likelihood of relapse in TNBC
HER2+ subtype has a similar response to TNBC
Bosch et al. / Cancer Treatment Reviews 36 (2010) 206–215
Basal-like and HER2+/ER subtypes are more sensitive to anthracycline-based neoadjuvant chemotherapy than luminal breast cancers. Patients that had pathologic complete response to chemotherapy had a good prognosis regardless of subtype. The poorer prognosis of basal-like and HER2+/ER breast cancers could be explained by a higher likelihood of relapse in those patients in whom pathologic complete response was not achieved.

12. Beyond brute force chemo: What are some potential treatment options for TNBC?
Current and developing therapies
Many in clinical trials
Most target proliferative pathways
Targets: General proliferation, surface molecules, secondary messengers

13. Potential Systemic Targets for TNBC

14. Platinum Agents
Platinum agents can bind to DNA and cause cross-linking to occur  cell death
Cisplatin, carboplatin and oxaplatin are approved for some types of cancers and are being studied as treatments for TNBC

15. PARP Inhibitors PARP: poly ADP ribose polymerase
Involved in DNA repair by detecting single-strand breaks
Can be activated in cells with damaged DNA
Several types of cancer are more dependent on PARP, so it can be a good therapeutic target
PARP inhibitors prevent breaks from being repaired, causing cell death.

16. Anti-EGFR EGFR is overexpressed in 45-70% of TNBC
Cetuximab is an anti-EGFR antibody used to treat metastatic cancer
Breast cancer patients with metastatic disease respond twice as well when Cetuximab is added
Other treatments include tyrosine kinase inhibitors (erlotinib, gefitinib)
Gefitinib is the only one currently approved for breast cancer, but the others are in clinical trials
Inhibits an important signaling pathway and provides a specific target!

17. Angiogenesis in Cancer
Angiogenesis: formation of new blood vessels.
Tumors need blood vessels to grow and spread.
Angiogenesis inhibitors prevent the formation of new blood vessels, thereby stopping or slowing the growth or spread of tumors.

18. Anti-Angiogenesis Bevacizumab (Avastin)
Monoclonal antibody to VEGF
Improves survival in breast cancer patients with combined with Taxol
Approved for metastatic breast cancer but benefit isn’t subtype specific – this has since been revoked because it slowed progression but didn’t extend length or quality of life and had many adverse effects
Metronomic chemotherapy: repeated, low, less than toxic doses can destroy endothelial cells and prevent angiogenesis, slowing tumor growth – works in clinical trials

19. Androgen Receptor
Nuclear receptor activated by binding testosterone or dihydrotestosterone
Closely related to PR
Expressed in 75% of breast cancer and 10-20% of TNBC
TNBC that express AR are molecularly similar to prostate cancer and could potentially be treated similarly.
Bicalutamine: anti-androgen used to treat prostate cancer
17-DMAG: semi-synthetic antibiotic derivative, has shown promise in clinical trials
Enzalutamide: androgen agonist used to treat prostate cancer; is in Phase II for TNBC

20. RTK Inhibitors Suninitib (Sutent) Imatinib (Gleevec)
Multiple-target RTK inhibitor
All PDGFRs and VEGFRs
KIT (CD17) which drives the majority of all GI stromal tumors & several others
Imatinib (Gleevec)
Prevents phosphorolation of BCR-Abl, inhibiting signaling pathways necessary for cancer cell growth
BCR-Abl: Exists only in cancer cells!
Worked in vitro; no effect on metastatic breast cancer patients in Phase II

21. Src Tyrosine Kinase Src is overexpressed in breast cancer
Dasatinib: multiple tyrosine kinase inhibitor approved for CML
Possible efficacy in breast cancer - small effect seen in Phase II
In vitro: basal breast cancer cells were more sensitive!
Several others in trials also seem to have promising preclinical activity

22. mTOR
Cell cycle regulator and a downstream effector in the PI3K/PTEN/AKT pathway
PTEN is often mutated in TNBC, leading to increased AKT and mTOR activation
Everolimus and temsirolimus block mTOR function and inhibit proliferation
Everolimus is approved for some types of cancers - currently in clinical trials for TNBC in combination with chemotherapy
Temsirolimus is approved for renal cell carcinoma and completed a Phase II trial with promising results

23. Other possible therapeutic options
Hsp90 (heat shock protein 90) – upregulated in response to stress signals; regulates and stabilizes many key proteins, including downstream targets of p53, PI3K, AKT and EGFR – can be recruited to ‘protect’ oncogenic proteins, leading to protein overexpression
HDAC (Histone deacetylase) – can effect epigenetics and cause re-expression of epigenetically silenced genes

24. Other ways to sensitize cells to chemotherapy
Inhibition of TGF-beta sensitizes to chemo
TRAIL: Lexatumumab (monoclonal antibody in clinical trials)
TRAIL controls proliferation & induces apoptosis
Chk1 (checkpoint kinase 1): involved in cell cycle control.
Inhibition sensitizes proliferating tumor cells to chemotherapies that damage DNA
TGFbeta: controls prolif in normal cells; not cancer cells.
Trail induces apoptosis

25. Mutations that Could be Targeted
p53 (75% of TNBC) – complex, so target downstream components of pathway
Myc (40% of TNBC)
Loss of retinoblastoma gene (20% of TNBC)
Mutation in BRCA1 or BCRA2 (15-20% of TNBC)
Rare:
PTEN
PIK3CA
Amplification of HER2
Amplification of FGFR2
RB: tumor suppressor

26. We need to get creative: changes in formulation
EndoTAG®-1: formulation of paclitaxel combined with neutral and positive lipids
Interacts with newly developing and negatively charged endothelial cells that are forming new blood vessels
Attacks the activated endothelial cells as they divide
Targets blood supply to tumors without affecting healthy tissue
Prevents angiogenesis and inhibits tumor growth!!

27. What’s in clinical trials now?
New compounds
New drug combinations or dosing regimens
New formulations
Interesting Current Clinical Trials
Re-expression of ER in Triple Negative Breast Cancers
Bevacizumab, Metronomic Chemotherapy (CM), Diet and Exercise After Preoperative Chemotherapy for Breast Cancer
Laboratory-Treated T Cells After Chemotherapy in Treating Women With Stage II or Stage III Breast Cancer Undergoing Surgery
Preoperative Clinical Trial of Sorafenib in Combination With Cisplatin Followed by Paclitaxel for Triple Negative (ER-, PR-, Her2-) Early Stage Breast Cancer
Clinicaltrials.gov

28. Recent news stories
March 18, Copper depletion shows early success in triple-negative breast cancer
April 8, 2013 – Paragazole (HDAC) excels in preclinical models of triple-negative breast cancer
April 12, Omega-3 Fatty Acids Slow Triple-Negative Breast Cancer Cell Proliferation
April 15, Nanodiamonds could improve effectiveness of breast cancer treatment

29. Outlook for now and future
Need targeted therapies, new formulations to be able to treat TNBC
Combination therapies will be necessary because tumors are heterogeneous and can change
Also need to attack tumors from all sides
Reaching complete remission and preventing recurrence are key

30. References A. Bosch et al. Cancer Treatment Reviews 36 (2010) 206–215
Cleator et al. Triple-negative breast cancer: therapeutic options. Lancet Oncol 2007; 8: 235–44
Pal et al. Triple negative breast cancer: unmet medical needs. Breast Cancer Res Treat (2011) 125:627–636
Crown et al. Emerging targeted therapies in triple-negative breast cancer. Annals of Oncology 23 (Supplement 6): vi56–vi65, 2012
Oncology (Williston Park) October ; 22(11): 1233–1243.
Hudis and Gianni. Triple-Negative Breast Cancer: An Unmet Medical Need. The Oncologist 2011, 16:1-11. doi: /theoncologist.2011-S1-01
Lisa A. Carey, E. Claire Dees, Lynda Sawyer, et al. Clin Cancer Res 2007;13:
Turner N et al. Targeting triple negative breast cancer: Is p53 the answer? Cancer Treat Rev (2013), /j.ctrv