1. About these slides SPEC – Short Presentation in Emerging Concepts Provided by the CAP as an aid to pathologists to facilitate discussion on the topic. Content has been reviewed by experts at the CAP, but does not necessarily reflect the official opinion of the College of American Pathologists. Non-CAP material with identified copyright source may only be copied or distributed under a license (permission) from the copyright holder, or under the doctrine of fair use. Version 1.0, rev. 12/31/2013

2. Emerging Concepts in Molecular Testing in Breast Cancer Short Presentation in Emerging Concepts (SPEC)

3. Is molecular/biological profiling the future of breast cancer diagnosis? The answer is YES! The role of molecular profiling in clinical practice is still evolving. What is the most practical, relevant, cost-effective, & broadly applicable ancillary testing that can help determine prognosis? o Can this approach be reliably used to guide the selection of beneficial treatment regimens? o Can some patients be spared the cost and morbidity of treatments that will be ineffective? o What is the level of evidence that the testing strategy can reliably answer these questions? o Do the molecular test results correlate with the patient’s clinical profile? The Pathology community has an opportunity to correlate molecular results and help inform treatment planning.

4. Decisions on systemic adjuvant therapy based on risk assessment: o Weigh background level - risk of recurrence against benefits & burdens of adjuvant therapy o Patient factors (clinically validated)  Age, menopausal status and co-morbidities o Tumor-related factors (clinically validated)  Tumor size (tumor burden), grade, LN, LVI Factors are robust prognostic markers, weaker in predicting for Rx response Are traditional approaches to making decisions about adjuvant treatment still useful?

5. How does breast cancer staging (TNM) influence prognosis? Tumor Size (pT)Nodal Stage (pN)TNM Staging TNM staging is used to help determine prognosis and risk of recurrence Staging affect s choice of therapy and treatment planning pT and pN reflect the patients tumor burden which is independently associate with prognosis T1 = < 2 cm T2 = 2.1 – 5 cm T3 = > 5 cm T4 = skin/chest wall invasion N0 = no lymph node involvement N1 = 1 – 3 positive nodes N2 = 4-9 positive nodes N3 = > 10 positive nodes Cancer 1989;63:181–187Cancer 1983;52:1551–1557

6. How does histologic grade influence prognosis? Histopathology. 1991;19(5):403-410, J Clin Pathol. 2002 Feb;55(2):88-92, J Clin Oncol. 2007;25(10):1239-46 Modified SBR Grade 3-5: Grade I - Well 6-7: Grade II - Moderate 8-9: Grade III – Poor Significantly correlates with DFS & OS HG1HG2 HG3

7. Treatment thresholds are based on disease biology, tumor characteristics and estimated risk for recurrence. What criteria are currently used to predict benefit from systemic adjuvant therapies? *ASCO-CAP Guidelines Type of TherapyCriterion Endocrine therapyAny ER staining by IHC (>1% ER+ invasive tumor)* Anti-HER2 therapyHER2 positive by IHC: circumferential membrane staining that is complete and intense, observed in a homogeneous and contiguous population and within >10% of the invasive tumor cells*. By ISH: HER2/CEP17 ≥ 2.0* Chemotherapy HER2+ disease = Chemo + HER2-targeted therapy Triple negative (ER-, PR- and HER2-) = Chemo ER+/HER2 negative = variable indication for Chemo, based on biology + risk

8. Treatment thresholds are based on disease biology, tumor characteristics and estimated risk for recurrence. What criteria are currently used to predict benefit from systemic adjuvant therapies? *ASCO-CAP Guidelines Type of TherapyCriterion Endocrine therapyAny ER staining by IHC (>1% ER+ invasive tumor)* Anti-HER2 therapyHER2 positive by IHC: circumferential membrane staining that is complete and intense, observed in a homogeneous and contiguous population and within >10% of the invasive tumor cells*. By ISH: HER2/CEP17 ≥ 2.0* Chemotherapy HER2+ disease = Chemo + HER2-targeted therapy Triple negative (ER-, PR- and HER2-) = Chemo ER+/HER2 negative = variable indication for Chemo, based on biology + risk

9. Which ER(+)/HER2(-) breast cancer patients are the most likely to benefit from chemotherapy? (St. Gallen's) Relative indication for chemotherapy Factors not useful for decision* Relative indication for endocrine therapy alone Pathologic Features ER & PRLow expression of ER & PR (or ER[+]/PR[-]) High expression of ER & PR Histologic gradeGrade 3Grade 2Grade 1 ProliferationHigh (Ki-67 >20%)Borderline (10-20%)Low (Ki-67 <10%) Nodal statusPositive (4 or more)Positive (1-3 nodes)Negative LVIPresence (especially extensive) +/-Absence Tumor size (pT)> 5 cm2.1 – 5 cm< 1-2 cm *Validated multigene assays may be an adjunct to high-quality pathologic phenotyping if doubt about the indications for chemotherapy persist after consideration of all pathologic factors Multigene assay*High risk scoreIntermediateLow risk score Goldhirsch Annals of Oncology, 20:1319-1329(2009)

10. New Approaches to an old problem State-of-the-art molecular technology can be used to analyze breast cancers samples at the DNA, RNA and/or protein levels. Molecular profiling in early breast cancer can be used to: o Refine breast cancer classification o Assess prognosis o Assess response to therapy Garber. Science. 2004;303:1754-1755. Can genomic profiling and multi-gene assays help determine prognosis and response to treatment in breast cancer?

11. What are the different ways we can look at breast cancer to understand prognosis? FeaturesLow Grade DiseaseHigh Grade Disease Histologic Grade (Morphology) DNA copy # changes (CGH Pattern) RNA expression (Gene Expression Profiling) Clinical SignificanceIndolent clinical courseAggressive clinical course +1q/-16q/simple changesrecurrent amplifications, and deletions

12. How does histologic grade correlate with gene expression? G1G3 Low GGI = Low Risk Recurrence High GGI = High Risk Recurrence J Clin Oncol. 2007;25(10):1239-46, Sotiriou, C. et al. J. Natl. Cancer Inst. 2006 98:262-272 G1G2G3 Histologic grade significantly correlates with prognosis G1 and G3 tumor show different gene expression profiles These profiles (genomic grade index) are associates with: - Low risk for recurrence (G1) - High risk for recurrence (G3)

13. Breast Cancer ER- ER+ HER2- Ki67 <14% ER+ HER2- Ki67>14% ER+ HER2+ ER- HER2+ ER/PR/HER2- CK5/6 +/- EGFR ER- PR- HER2- ER+ HER2+ Luminal B Luminal HER2 Luminal A HER2 Enriched Basal-Like TNBC Non-Basal Prognostic Significance How do pathologic features correlate with molecular classification? Perou, Sorlie et al. 2001, 2003. Cheng, JNCI 2009.

14. What factors are helpful to predict for chemotherapy sensitivity/benefit in breast cancer? Factors in favor of selecting adjuvant chemotherapy Clinical/Pathologic features –ER negative –Ductal histology –Grade 3 –High proliferation Tumor biology/molecular –Basal-like (chemo) and HER2 positive (c hemo + HER2 targeted therapy) –High risk MammaPrint®, Oncotype DX® or GGI Factors against selecting adjuvant chemotherapy Clinical/Pathologic features –ER positive (high expression) –Lobular histology (classic) –Grade 1 –Low proliferation Tumor biology/molecular –Luminal A type –Low risk MammaPrint®, Oncotype DX® or GGI Nat Rev Clin Oncol, 8;272-279(2011)

15. Molecular Classification Luminal ALuminal BHER2+‘Basal-Like’ 70 gene 76 gene Recurrence score Genomic grade Gene-Expression Prognostic Signatures Quantitative measurement of proliferation and differentiation genes Well differentiated/low proliferation Low Grade Good Prognosis Poorly differentiated/high proliferation High Grade Poor Prognosis Genes associated with tumor differentiation & cell cycle drive the prognostic power of the intrinsic molecular classification and several gene expression signatures Sotiriou et al. NEJM 2009;360:790-800. What approaches have been used to classify breast cancer based on gene expression data?

16. High Tumor Proliferation Genomic Grade Index 70 gene signature 76 gene signature 21 gene recurrence scoreIntrinsic Molecular Classification Intrinsic Biologic Properties of Breast Tumor Aggressive Clinical Course Poor Prognosis More Responsive to Chemo  Proliferation genes are a common driving force in prognostic signatures.  Tumors with high proliferation are more aggressive and have a better chemo response. What intrinsic properties of breast cancer are most important for understanding clinical course?

17. What methods do pathologists have to assess proliferation in clinical breast cancer samples? Counting mitotic figures (mitotic activity index, MAI) o Part of breast cancer histologic grading o Careful MAI assessment predicts prognosis, chemo benefit Ki67 expressed in all phases of the cell cycle other than the (G0) phase o High Ki67, associated with worse outcomes, chemo benefit in some studies o Limitations - enormous variation in analytical practice, lack of standardization for scoring o Need for comprehensive standardization on pre-analytical and analytical assessment, interpretation and scoring of Ki67 Phosphohistone H3 (PHH3) marker of cells in G2 & M Phase of cell cycle Standardization of current methods and/or better ways of assessing proliferation in routine clinical samples are needed Breast Cancer Res Treat (2009) 115:241–254, JNCI Vol. 103, Issue 22, 2011, Breast Cancer Res Treat (2009) 114:39–45

18. Molecular results must correlate with clinical & pathologic features for the patient’s tumor. Discordance with clinical or pathologic findings must be reconciled prior to treatment decisions. Pathologists provide understandable, comprehensive reports and are aware of the clinical implications for each of the reporting elements that they provide to the treatment team. Pathologists are important members of the patient care team helping to integrate these results. Tumor Burden Clinical Presentation Tumor Grade (Differentiation) and Proliferation Molecular Profile How can profiling/molecular testing be integrated into treatment planning for breast cancer?

19. What elements are reported on breast cancer at ? Reporting ElementsClinical Significance Histologic gradeCorrelates with clinical course and potential benefit from systemic therapy Tumor SizeMeasurement of tumor burden, correlates with prognosis and likelihood of recurrence LVICorrelates with lymph node mets, local and distant recurrence Nodal StatusMeasurement of tumor burden, correlates with prognosis and likelihood of distant recurrence HER2Correlates with aggressive disease and benefit from chemo and targeted- therapy ER/PRCorrelates with benefit from endocrine therapy ProliferationCorrelates with clinical course and benefit from systemic therapy (most useful for ER+) Margin statusCorrelates with likelihood of local recurrence Multigene assaysCorrelates with prognosis and potentially systemic treatments benefit Any molecular test or multi-gene assay regardless of whether it is done in house or not needs to be correlated with the tumor morphology and clinical features.

20. Selected Resources Tang P, Skinner KA, Hicks DG. Molecular classification of breast carcinomas by immunohistochemical analysis: are we ready? Diagn Mol Pathol. 2009;18(3):125-32. Sotiriou C, Pusztai L. Gene-expression signatures in breast cancer. N Engl J Med. 2009;360(8):790-800. Allison KH. Molecular pathology of breast cancer: what a pathologist needs to know. Am J Clin Pathol. 2012;138(6):770-80.

21. Additional Free Resource for CAP Members NOTE: please remove this page before presenting. CAP Member Exclusive: CAP Pathology Resource Guides Focused on a specific hot-topic technology, these comprehensive guides highlights current resources, select journal articles, as well as CAP and non-CAP educational opportunities. And don’t miss the “Insights From Early Adopters” section in each guide to gain perspective from pioneering colleagues. AVAILABLE NOW: Molecular Pathology (single gene test, small panel) Genomic Analysis (large panel, exome, genome) Learn more: go to cap.org and type Pathology Resource Guides in the “search” field located at the top of your screen. “Extremely well done, of high practical and educational value.” “An outstanding overview of basic materials, including the technology and links to a number of individuals and centers that can assist.”