Case 6 A 49 year old female was found to have a 1.3 cm spiculated mass on screening mammogram Ultrasound revealed a 1.2 cm hypoechoic mass with posterior shadowing and irregular margins Core needle biopsy revealed a 1.4 cm, grade 2, invasive ductal carcinoma that was ER+, PR- and HER2- Subsequent excision confirmed the tumor size and grade; No LVI was identified A sentinel lymph node was negative The medical oncologist requests OncotypeDX

Case 6 A 49 year old female was found to have a 1.3 cm spiculated mass on screening mammogram Ultrasound revealed a 1.2 cm hypoechoic mass with posterior shadowing and irregular margins Core needle biopsy revealed a 1.4 cm, grade 2, invasive ductal carcinoma that was ER+, PR- and HER2- Subsequent excision confirmed the tumor size and grade; No LVI was identified A sentinel lymph node was negative The medical oncologist requests OncotypeDX

“Luminal B-like Tumor” Case 6 ER strongly positive; 90% of tumor cell nuclei stained PR negative HER2 negative “Luminal B-like Tumor” Caveat: “Attempts to reproduce the intrinsic subtype distinction between Luminal A-like and Luminal B-like using conventional pathology have proved impractical”- St. Gallen, 2015

Luminal B Breast Carcinoma ~10% of breast cancers ER positive Tend to be higher grade and/or have a higher proliferation index May overexpress HER2 (Luminal B, HER2+) High expression of hormone receptors and associated genes Respond to endocrine and chemotherapy Prognosis not as good as for Luminal A Sorlie, 2001

Immunophenotyping to Approximate Molecular Subtype Maisonneuve, BCR, 2014 Coates, Ann Oncol, 2015 Luminal A ER+, HER2-, Ki-67<14% or Ki-67 intermediate (14-19%) and PR>20%, Luminal B ER+, HER2- Ki-67 intermediate (*14-19%) and PR- or low (<20%) or Ki-67>20% HR+, HER2+ HER2 enriched ER-, PR-, HER2+ Basal-like ER-, PR-, HER2-, [CK5/6+ and/or EGFR+] *St. Gallen, 2015 considers PR 20-29% to be intermediate

Luminal B Breast Carcinoma < Ades, JCO, 2014

Molecular Classification Implications for Prognosis

Molecular Classification 2016 Breast Cancer Estrogen Receptor Negative Cancers Positive Cancers Basal-like ER, PR, HER2 negative HER2 Enriched HER2+ ER/PR absent Luminal B-like LB-HER2-: ER+/HER2-, Either Ki-67 high or Ki-67 intermediate and PR-/low LB-HER2+: ER+/ HER2+ Any Ki-67, Any PR Luminal A-like ER+, HER2- and Ki-67 low or Ki-67 intermediate and PR high

Molecular classification has prognostic significance Ciriello, BCRT, 2013 Sorlie, 2003

Commercially Available Multigene Signatures Van de Vijver, 2014

OncotypeDx (Genomic Health, Inc.) <18 Low 18-31 Intermediate RS = +0.47 x HER2 group score -0.34 x ER group score +1.04 x proliferation group score +0.10 x invasion group score +0.05 x CD68 -0.08 x GSMT1 -0.07 x BAG1 <18 Low 18-31 Intermediate >31 High NEJM 2004;351:2817

Recurrence Score and Prognosis in ER+, N- Breast Cancer Paik, 2004

Drukker, BCRT, 2014 Expression signature consisting of 70 genes identified good and poor prognosis groups among both N- and N+ patients Better than standard prognostic systems based on clinical and histologic features (e.g., St. Gallen, NIH) MammaPrint (Agendia) NEJM, 2002

Prognostic value independent of: Nodal status Size Grade ER status J Clin Oncol 2009 Prognostic value independent of: Nodal status Size Grade ER status Predicted benefit from neoadjuvant chemotherapy PAM50 Assay

Which Expression Signature is Best? Fan, NEJM, 2006 Five different gene-expression based models compared among a single set of 295 samples High level of concordance in outcome prediction But, very little overlap in genes

Which Expression Signature is Best? Fan, NEJM, 2006 OncotypeDX Mammaprint Wound Healing

Proliferation genes are the common driving force in all prognostic signatures Factors associated with tumor burden (size, nodal status) remain independently associated with prognosis Breast Cancer Res 2008

Comparison of 47 published breast cancer signatures with 1,000 randomly generated signatures >90% of signatures predicted breast cancer outcome; 60% of random ones performed better than original signatures Prognostic signatures do not identify specific mechanisms, rather all signatures, even random ones, are proliferation related and able to discriminate prognosis PLoS, 2011

Most can be performed on FFPE Relevance to Clinical Practice Are Expression Signatures Ready for Routine Clinical Use? Most can be performed on FFPE Almost all available data from retrospective analyses Prognostic value time dependent (reduced between 5-10 years) Cost

Relevance to Clinical Practice Which patients? All signatures more useful for assessing prognosis in ER+ cancers (i.e., luminal) and are of little value in ER- cancers (i.e., HER2 and basal molecular types)

Relevance to Clinical Practice Is this approach really better than using a combination of clinical and pathologic factors supplemented by appropriate biomarkers detected by IHC (e.g., ER, PR, HER2 and Ki67)?

IHC4 Score Cuzick, JCO, 2011 1125 pts with ER+ breast cancer in the TransATAC trial ER, PR, HER2 and Ki67 assessed by IHC Combined “IHC4 Score” provided similar prognostic information as OncotypeDX Recurrence Score

Surrogate Histologic and IHC Markers in Clinical Practice-Ki67 Proliferation markers used to differentiate Luminal A from Luminal B Unlike ER and HER2 which show bimodal distribution with clear cutpoints, proliferation determined by several genes with continuous distribution

Surrogate Histologic and IHC Markers in Clinical Practice-Ki67 Tumor grade most widely used as a surrogate for proliferation Ki67 most widely used proliferation marker No consensus on cutpoint or method of counting ?Better to utilize prognostic signature in this scenario

More patients scored as high risk and fewer as intermediate risk JCO, 2013 PAM50 Risk of Recurrence (ROR) Score provided more prognostic information in endocrine-treated, ER+, node- patients than OncotypeDx recurrence score, especially in HER2- group More patients scored as high risk and fewer as intermediate risk

JCO, 2013

Validation of Nanostring’s Prosigna Assay PAM50 ROR Nielsen, Clin Cancer Research, 2014 Wallden, BMC Med Genomics, 2015 The analytical performance of the Prosigna assay based on PAM50 has been validated using FFPE across multiple laboratories Accurate prediction of risk of distant recurrence among women with ER+ breast cancer treated with 5 yrs of tamoxifen (prognostic beyond 5 years) Able to classify tumors according to intrinsic subtype

Alvarado, Adv Ther, 2015

Alvarado, Adv Ther, 2015

Multigene Signatures and Predictive Factors

Recurrence Score and Chemotherapy Benefit In ER+, N- Breast Cancer Paik, 2006 Low Int High

Being used clinically with increasing frequency 2015 Being used clinically with increasing frequency OncotypeDX RS used most often to identify patients with ER+ breast cancer who may safely be spared cytotoxic therapy (i.e., those with low recurrence score)

Current Clinical Status of Expression Signatures For Selecting Treatment “For patients with ER+ early breast cancer the benefits of OncotypeDX outweigh the acquisition costs” (Rouzier, BCRT, 2013) “It is one step more toward precision” (Hudis, NEJM, 2015)

Where are we today? ER, PR and HER2 status are the major drivers of clinical decision making regarding the type of systemic therapy OncotypeDx used in some patients with ER+ breast cancer to determine the need for chemotherapy

Where are we today? Targeted sequencing for genomic alterations/mutations in patients with metastatic disease to determine eligibility for clinical trials (e.g. for PI3 kinase inhibitors)

Signaling Pathways Under Blockade in Luminal Cancers Ades, JCO, 2014

Chemotherapy Benefit? Three prospective randomized trials-MINDACT, TAILORx and RxPONDER- are testing the usefulness of gene signatures in predicting benefit from adjuvant chemotherapy in patients with ER+ breast cancer Results anticipated 2015-2017

Conclusions Molecular pathology has: Furthered our understanding of breast cancer molecular mechanisms Provided insight into the development of targeted therapies A role in determining prognosis and predicting response to therapy Incorporation of expression signatures into clinical practice is here

Lesions that may mimic Invasive Carcinoma Conclusions Lesions that may mimic Invasive Carcinoma For optimal patient care, we should be thinking about breast cancer according to the molecular subtypes (or the clinicopathologic correlate) with the associated prognostic and therapeutic implications