1. 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

4. 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

5. IHC in Molecular Classification and ER Testing

6. 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

7. 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)

8. Livasy 2005, Mod Pathol

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

12. 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

13. 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

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

15. 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

16. BASAL-
LIKE
TRIPLE
NEGATIVE
~70-80%

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

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

19. 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

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

21. 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

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

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

24. Clinicopathologic Subtype within Molecular Subtype
Prat, Mol Oncol, 2011

25. ER

26. 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

27. 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:
©2006 by American Society of Clinical Oncology

28. 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)

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

30. 2010

31. 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

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

33. 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

34. 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

35. 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

36. 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

37. 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

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

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

40. 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

41. 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

42. 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

43. 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

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

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

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

47. 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

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

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

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

51. 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

52. 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

53. 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

54. 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

55. 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

56. 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

57. 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

58. 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

59. 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

60. 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

61. 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)

62. 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