Case 2 A 34 year old female with a strong family history of breast cancer presented with a palpable breast mass The mass presented in the interval between alternate biannual MRI and mammogram screens An ultrasound-guided core needle biopsy was performed Images are from the subsequent excision specimen

Case 2 Invasive ductal carcinoma, grade 3 ER negative (<1% nuclei staining) PR negative (0% nuclei staining) HER2 negative (0 by IHC; no amplification by FISH) Triple Negative or Basal-like Carcinoma

IHC in Molecular Classification and ER Testing

Basal-Like Carcinoma Subtype of invasive breast cancer identified through gene expression profiling studies Express genes characteristic of basal epithelial cells Comprise ~ 15% of invasive breast cancers Sorlie, 2001

Histology of Basal-Like Cancers Identified By Expression Profiling Livasy, Mod Pathol, 2005 Histologic grade 3 (100%) Solid architecture No tubule formation, high density of cells with no intervening stroma Pushing border (61%) Stromal lymphocytic infiltrate (56%) High mitotic rate (100%) Geographic zones of necrosis (74%) Medullary-like features (Central fibrotic/acellular zone) (Little or no associated DCIS)

Livasy 2005, Mod Pathol

Here is another example of a poorly differentiated carcinoma, with a central fibrotic core and a solid growth pattern of tumor cells

Basal-like cancers as defined by expression profiling: Sorlie, 2003 Basal-like cancers as defined by expression profiling: Poor prognosis Often seen in women with BRCA1 mutations Preponderance of African-American women

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

Basal-like Breast Carcinoma vs. Triple Negative Breast Carcinoma A word on terminology Basal-like Breast Carcinoma vs. Triple Negative Breast Carcinoma

Clinicopathologic definition: Surrogate Definitions of Intrinsic Subtypes Basal-like Carcinoma Goldhirsch, Ann Onc, 2011 Intrinsic subtype: “Basal-like” Clinicopathologic definition: Triple negative (ductal) ER and PR absent, HER2 negative

BASAL- LIKE TRIPLE NEGATIVE ~70-80%

Histologic Features of Basal-Like Cancers Invasive Ductal Medullary Adenoid Cystic Metaplastic

Are They “Basal-Like”? Weigelt, J Pathol, 2008

Refining the Categorization of Basal-like Cancers Hennessy, Cancer Research, 2009 Prat, Breast Cancer Research, 2010 Metaplastic breast carcinomas are molecularly distinct group Most closely related to “claudin-low” subgroup Claudin-low group characterized by loss of a group of genes encoding cell-cell adhesion Also express high levels of stem cell markers Histologically “spindloid” morphology

LMWK and ER related genes Markers of EMT (inc claudin) Markers of stem cell related genes Prat BCR, 2010

Mesenchymal stem-like TNBC Subtypes cisplatin Basal-like 1 Basal-like 2 Immunomodulatory Mesenchymal-like Mesenchymal stem-like AR antagonists Luminal AR Lehmann, JCI, 2011

Comprehensive Genome Analysis Identifies Novel Subtypes and Targets in TNBC Burstein, CCR, 2015 1. LAR 2. MES 3. BLIS 4. BLIA

Activation of different signaling pathways J Pathol, 2014 Activation of different signaling pathways Therapeutic implications

Clinicopathologic Subtype within Molecular Subtype Prat, Mol Oncol, 2011

ER

ER Interpretation/Scoring >10% = positive Fewer positives Pts potentially denied therapy >1% = positive End up with a lot more positives! Pts potentially treated with little benefit

SP1 8% more sensitive than 1D5 using DCC as standard SP1 Sensitivity of Ab used or antigen retrieval method can change a test result from negative to positive . 1D5 SP1 8% more sensitive than 1D5 using DCC as standard SP1 Cheang M C et al. JCO 2006;24:5637-5644 ©2006 by American Society of Clinical Oncology

Estrogen Receptor IHC Issues Multiple sources of variability exist in any given laboratory -pre-analytic variables (e.g. fixation times) -choice of antibody -antigen retrieval techniques -use of controls -interpretation/scoring (?cut points too high)

Influence of Fixation Time Goldstein, Am J Clin Pathol, 2003

2010

GOAL Improve accuracy of hormone receptor testing and the utility of ER and PR as prognostic and predictive markers for assessing in situ and invasive breast carcinomas Standardization

Accurate measurement of ER is critical for the care of all breast cancer patients

Estrogen Receptor in Breast Cancer ER is a weak prognostic factor But a strong predictive factor Thus women with ER+ cancers have a strong likelihood for responding to hormonal therapies

Quantitation of ER IHC qualitative test Semi-quantitative at best Sensitivity of antibody used or antigen retrieval method can change a test result from negative to positive

Dichotomization of ER The need for quantification of ER at this time is uncertain (AM Gown, Mod Pathol, 2008) If the question is whether to treat or not to treat, dichotomization of ER seems reasonable

Dichotomization of ER NIH Consensus Statement on Adjuvant Therapy for Breast Cancer (2000) Any degree of ER nuclear staining detected by IHC should be considered a positive result, thus rendering the patient eligible for endocrine therapy

Quantification of ER Why quantify? “The percentage of stained tumor cells may provide valuable predictive and prognostic information to inform treatment strategies” ASCO/CAP Guidelines, 2010

ER Level and Disease-free Survival Allred score of 3 equivalent to 1% of nuclei positive Harvey J M et al. JCO 1999;17:1474-1474 ©1999 by American Society of Clinical Oncology

Allred Score Distribution Harvey, 1999 584 401 370 320 Number of cases 190 117 Allred score n=1982

Categories of Endocrine Responsiveness Goldhirsch, St Categories of Endocrine Responsiveness Goldhirsch, St. Gallen Conference 2007, Ann Oncol Highly endocrine responsive: Tumors express high levels of both HRs in the majority of cells Incompletely endocrine responsive: Some expression of HRs but at lower levels or lacking either ER or PR Endocrine non-responsive: Tumors having no detectable expression of steroid hormone receptors

Categories of Endocrine Responsiveness Goldhirsch, St Categories of Endocrine Responsiveness Goldhirsch, St. Gallen Conference 2007, Ann Oncol Some degree of quantitation is needed to distinguish the “highly endocrine responsive” from the “incompletely endocrine responsive” groups

Quantification of ER All positively associated with ER levels Overall survival Disease-free survival Recurrence/relapse-free survival 5 year- survival Response to endocrine therapy Time to recurrence All positively associated with ER levels Elledge RM, 2000 In J Cancer Dowsett M, 2008, JCO Cowen PN, 1990, Histopathology Stendahl M, 2006, Clin Cancer Res Esteban JM, 1994, J Cell Biochem Suppl Yamashita H, 2006, Breast Cancer

Does IHC Permit Reliable Quantification of ER? Current IHC methods utilize highly sensitive antibodies and detection systems and often employ signal enhancement Dichotomization of Results

Allred Score Distribution Collins, 2005 661 Number of cases 157 3 4 Allred score n=825

Allred Score Distribution Badve, 2008 369 236 Number of cases 76 37 33 27 Allred score n=778

% Distribution Zhang, 2014 n=1,700 Number of cases % ER nuclear positivity n=1,700

Quantification of ER We know from LBA days that ER in breast cancer is a continuous variable ER is not biologically bimodal ?Need for alternative methodologies

ER by RT-PCR Badve, et al, 2008 Number of cases RT-PCR score 317 143 127 87 51 31 20 RT-PCR score

Comparison of ER IHC, Gene Signature Score and mRNA Expression Blue=0% Green=1-9% Purple=10% Gold=>10% Iwamoto, JCO, 2012

% Distribution Iwamoto, 2012 Number of cases % ER nuclear positivity N=465

Low ER+ Breast Cancer; Is This a Distinct Group? Gloyeske, AJSP, 2014 Evaluated 49 cases of low ER+/HER2- Often grade 3 (92%), with sheetlike growth (71%), intratumoral lymphocytes (59%) and necrosis (45%) 80% tumors had ki-67 index >50% 94% PR-negative 33% achieved pCR with neoadjuvant chemotherapy

Low ER+ Breast Cancer Iwamoto, JCO, 2012 A minority of 1-9% ER IHC-positive tumors show features of ER+ tumors and are potentially endocrine-sensitive Most show features of molecular basal-like, ER negative tumors Likely best treated with chemotherapy and adjuvant endocrine therapy

22 tumors with 1-9% ER expression By gene expression profiling: None luminal 16 basal-like (73%) 6 HER2-E (27%) Ann Surg Oncol, 2013

Of 1,557 breast cancers, only 65 cases with borderline ER (4.1%) Defining Breast Cancer Intrinsic Subtypes by Quantitative Receptor Expression Cheang, The Oncologist, 2015 Of 1,557 breast cancers, only 65 cases with borderline ER (4.1%) Supports ASCO/CAP definition of <1% for ER negativity

66% if borderline cases included among TNBC Defining Breast Cancer Intrinsic Subtypes by Quantitative Receptor Expression Cheang, The Oncologist, 2015 Of 1,557 breast cancers, only 65 cases with borderline ER (4.1%) Supports ASCO/CAP definition of <1% for ER negativity 66% if borderline cases included among TNBC

Receptor Status # # (%) BRCA+ ER low pos 22 7 (32%) ER neg 122 Germline BRCA Mutations in Patients with ER low positive (<10%), PR Negative, HER2 negative tumors Sanford, ASCO Breast 2014 Receptor Status # # (%) BRCA+ ER low pos 22 7 (32%) ER neg 122 33 (27%) p=0.65

2.6% of tumors ER borderline DRFS RFS OS 2.6% of tumors ER borderline (1-9%) Endocrine Rx No endocrine Rx Ann Oncol, 2014

Current Recommendations Reporting of Results Percentage/proportion of positive tumor cells should be recorded Intensity of staining should be recorded: weak, moderate or strong An interpretation should be provided

Validated IHC Assay for ER All IBCs and DCIS Validated IHC Assay for ER <1% cells = Negative Expect 20%-30% overall Confirm/Retest if: Low grade Lobular Tubular Mucinous No Endocrine Therapy >1% cells = Positive Expect 70%-80% overall Quantification Endocrine Therapy ASCO/CAP, 2010 NCCN, 2009

Validated IHC Assay for ER Confirm/Retest on excision All IBCs and DCIS Testing done on CNB Validated IHC Assay for ER >1% cells = Positive Expect 70%-80% overall Quantification Endocrine Therapy <1% cells = Negative Expect 20%-30% overall Confirm/Retest on excision No Endocrine Therapy BIDMC, 2016

Lesions that may mimic Invasive Carcinoma Conclusions Lesions that may mimic Invasive Carcinoma Basal-like carcinomas are a heterogeneous group of tumors Low ER positivity should be reported to allow for the potential of endocrine therapy in combination with chemotherapy where appropriate (although ?utility of endocrine therapy in this group)

Conclusions The combination of highly sensitive ER assays and the low threshold for ER+ is likely resulting in the categorization as ER+ some breast cancers that are biologically and clinically more like ER-, or at least of “indeterminate” subtype ?Should reflex PAM50 testing be performed in low ER+ cases to clarify underlying biology