1. GENE EXPRESSION PROFILING IN BREAST CARCINOMA Dr Partha Ghosh 2 nd year PGT, General Surgery N.R.S Medical college

2. What is gene expression profiling? Cancer is a disease characterized by uncontrolled cell growth and proliferation. For cancer to develop, genes regulating cell growth and differentiation must be altered; these mutations are then maintained through subsequent cell divisions and are thus present in all cancerous cells. Gene expression profiling is a technique used in molecular biology to query the expression of thousands of genes simultaneously. In the context of cancer, gene expression profiling has been used to more accurately classify tumours. The information derived from gene expression profiling often has an impact on predicting the patient’s clinical outcome.

3. Tumor biology IHC mRNA profiling Histology TNM Staging

4. 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  TYPE A:ER+, PR+,HER-2-  TYPE B:ER+,PR+,HER-2+  Can use hormonal therapy  Less aggressive ◦ Basal: derived from myoepithelial cells  ER-, PR-,HER-2-,ck 5/6+ or HER -1+  No specific target for therapies  More aggressive ◦ HER2-enriched  More aggressive Luminal A Luminal B Claudin-Low HER2-enriched Basal

5. Luminal and Basal Characteristics Basal Low ER Low HER2 High CK5/6 c-KIT higher High EGFR High p53 mutation High p53 protein High cyclin E Very high vimentin Luminal High ER Higher HER2 Low CK5/6 Low c-KIT Low EGFR Low p53 mutation Low p53 protein Low cyclin E Low vimentin Basement membrane Myoepithelial Cells  Basal Luminal Cells  Luminal

6. What is HER 2/neu Other names ◦ Receptor tyrosine-protein kinase erbB-2 ◦ CD340 ◦ proto-oncogene Neu ◦ ERBB2 (human) Located in chromosome 17 (17q12) Rodent glioblastoma cell line a neural tumour

7. Cont…. Plasma membrane bound receptor tyrosine kinase Positive in 15-30% cases of breast carcinoma Associated with increase disease recurrence and poor prognosis Tested by- ◦ Immunohistochemistry ◦ FISH ◦ Serum HER2 by ELISA- Trastuzumab response

8. Cont… Drugs targeting HER2 Trastuzumab- p27 halts cell proliferaton Pertuzumab- prevent dimerisation of HER2 and HER3 NeuVax- Direct killer T cells to destroy HER2 +ve cancer cells

9. Tamoxifen resistant breast ca The expression of HER2 is regulated by signaling through estrogen receptors. Normally tamoxifen down regulate HER2 Ratio of coactivator AIB-3 exceed that of corepressor PAX2 HER2 expression in presence of tamoxifen

10. Algorithm for breast cancer subtypes All cases ER - PR - ER+ or PR + HER2 - HER2 + HER2 - EGFR - CK5/6 – EGFR + or CK5/6 + Unclassified Basal LikeLuminal B Luminal A HER2+/ER-

11. Luminal A Luminal B HER2+ Basal-like Intrinsic Breast Cancer Subtypes described by Perou et al. Express ↑ amounts Of luminal cyto- Keratins & genetic Markers of luminal Epithelial cells of Normal tissue Express ↑ levels of EGFR, c-kit, & growth factors like hepatocyte growth factor and IGF

12. Mammaprint: Development of the 70- Gene Signature  DNA microarray analysis of 78 breast primary tumors (untreated)  Pts were <55 years of age with T1-2/N0 disease  Pts selected based on outcome: Distant metastases within 5 years  Statistical analysis, “supervised classification,” identified 231 genes correlated with disease outcome  Top 70 genes selected  Genes that regulate cell cycle, invasion, metastasis, & angiogenesis  Patients categorized as “good prognosis” or “poor prognosis.”  Found to be a better predictor of distant metastases within 5 years than all clinical variables in this study  Odds ratio (distant metastases): poor to good prognosis groups = 15

13. Does 70-gene Signature have Independent Prognostic Value?  Gene signature adds independent prognostic information to that provided by various risk classifications  The signature remained a statistically significant prognostic factor for time to distant metastases & OS even after adjustment for various risk classifications (HR 2.15 & 2.15, respectively)

14. The 21-gene Recurrence Score: Oncotype DX

15. Oncotype Dx: The 21-Gene Assay Designed to quantify the risk of distant recurrence in patients with LN(-), ER(+) tumors receiving tamoxifen RT-PCR was used to quantify gene expression from fixed, parafin-embedded tumor tissue 250 candidate genes selected based on published literature, genomic databases, & experiments based on DNA arrays on fresh-frozen tissue Analyzed data from 3 independent studies (447 patients) including tamoxifen-only arm of NSABP trial B-20 to test relation b/w 250 genes and recurrence of breast cancer From these studies, 16 genes (+5 reference genes) were selected that correlated with proliferation and endocrine response

16. Levels of Gene Expression Determine Recurrence Score 21-gene assay = 16 outcome-related genes + 5 reference genes Higher expression levels of “favorable” genes = ↓ RS Higher expression levels of “unfavorable” genes = ↑ RS A risk score is calculated from 0 -100 Cutoff points chosen based on Results of NSABP trial B-20

17. Prospective Validation of Oncotype DX:TAILORX Trial Low RS: Hormonal Therapy High RS: Chemo + Hormonal Therapy Hormonal Therapy Chemo + Hormonal 11,248 ER+/LN- patients

18. TN VS BASAL Subtypes Triple negative ◦ ER,PR,HER2 receptor negative Basal Subtypes ◦ ER,PR,HER2 receptor negative ◦ Over expression of breast basal cell epithelium gene ◦ EGFR+,CK5/6+

19. Basal like subtypes of breast cancer No proven therapeutic target ER,PR negative-cannot use tamoxifen or anti estrogen HER2 negative-cannot use Herceptin

20. Targeted therapy ER + ER- Hormonal Herceptin EGFR BRCA 1 C-kit Therapy Geftinib, erlotinib, DNA damage Imatinib lapatinib PARP inh Luminal ALuminal B HER 2 + Basal Like

21. Potential treatment options in TNBC

22. 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 TAC: docetaxel, doxorubicin, and cyclophosphamide every 3 weeks for 6 cycles  Dense dosing is better

23. 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

24. 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

25. 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!

26. 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.

27. 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

28. 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

29. RTK Inhibitors Suninitib (Sutent) ◦ 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

30. 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

31. 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

32. Conclusions Gene signatures augment current clinicopathological variables in assessing risk of recurrence Gene expression profiles may be both prognostic and predictive for patients with early breast cancer NCCN guidelines suggest that Oncotype DX is an option for risk evaluation in 0.6-1 cm tumors with unfavorable characteristics or in >1 cm LN-, ER+/HER2 negative tumors

33. NCCN guidelines include Oncotype DX ® testing in the treatment-decision pathway for node-negative and micrometastatic disease 33 Tumor 0.6-1.0 cm, moderately or poorly differentiated, intermediate or high grade, or vascular invasion Tumor > 1 cm with favorable or unfavorable pathologic features Tumor 0.6-1.0 cm, moderately or poorly differentiated, intermediate or high grade, or vascular invasion Tumor > 1 cm with favorable or unfavorable pathologic features Consider Oncotyp e DX Hormone receptor-positive, HER2-negative disease pT1, pT2, or pT3 and pN1mi No test RS < 18 RS 18- 30 RS ≥ 31 Adjuvant endocrine therapy ± adjuvant chemotherapy Adjuvant endocrine therapy endocrine therapy ± adjuvant chemotherapy Adjuvant endocrine therapy + adjuvant chemotherapy

34. THANK YOU