1. The combination of SMMHC and p63 is ideal for marking myoepithelial cells and assessing features such as stromal invasion and differentiation of in situ versus invasive lesions. 2. Positive 34βE12 and CK5/6 staining within the proliferative component indicates atypical papilloma rather than papilloma with DCIS. 3. Intracystic papillary carcinoma shows positive staining by Collagen IV around ducts, distinguishing it from solid papillary carcinoma.

1. IMMUNOHISTOCHEMISTRY IN BREAST
LESIONS
Dr. Ashish Jawarkar, MD

2. APPLICATIONS
OF IHC IN
BREAST
LESIONS
1. Myoepithelial cell staining and assessment of stromal
invasion
2. Differentiating papillary lesions
3. Tumor type identification (Ductal vs lobular)
4. Role in Paget’s disease
5. Detection of lymphatic space invasion
6. Sentinel lymph node evaluation
7. Identifying systemic metastasis of breast carcinoma
8. Fibroepithelial tumors
9. Theranostic applications

3. Myoepithelial
Markers
1

4. Myoepithelial
cells
Proliferative vs
malignant
In situ vs invasive
Pseudo invasive
(adenosis, radial scar,
sclerosing papillary
tumor) vs invasive

5. IHC profile of myoepithelial cells

6. Case 1
35/F with mass in left breast

7. Case 1
35/F with mass in left breast
SMMHC/calponin

8. Case 1
35/F with mass in left breast
p63
Most specific, present in nucleus

9. Problems with SMA
• Stains myofibroblasts, may give
false assurance of presence of
myoepithelial cells in dcis vs in situ
• In DCIS there is periductal stromal
desmoplasia

10. Problems with SMA
• SMMHC specifically stains MEC and
not myofibroblasts

11. Ideal panel Combination of SMMHC
and p63

12. Case 2
Breast core biopsy demonstrating
abundant intra and peritumoral
lymphocytic infiltrates
AE1/AE3
p63

13. Internal
controls
For SMMHC and calponin

14. Pitfalls of p63 and
SMMHC
• A p63 stain demonstrates apparent
gaps in staining around the luminal
epithelium of a duct within a radial
scar
• The same duct shows intense
continuous staining with smooth
muscle myosin heavy chain, but
myofibroblastic cells in the
background are also positive
• A p63 stain on core biopsy shows
staining of a few tumor cells
• A rare example of diffuse p63
staining of tumor cells

15. Caveat – DCIS
with papillary
config
Approximately 5% DCIS with a
papillary configuration may not
show myoepithelial cells
p63 negSMMHC neg• Collagen type IV
demonstrates strong
continuous staining around
tumor nests, confirming the
in situ nature of the lesion

16. Benign sclerosing lesions vs malignancy

17. Case 3
42/F vague lump in right breast
Sclerosing adenosis

18. Case 4
28/F with vague breast lumpRadial scar

19. Case 5
Cribriform growth pattern – In situ or invasive?

20. Caveat
SMMHC

21. PAPILLARY
LESIONS
2

22. PAPILLARY
LESIONS
• Papilloma
• Atypical papilloma (papilloma with ADH)
• Papilloma with DCIS
• Papillary DCIS (Encapsulated papillary carcinoma)
• Solid papillary carcinoma

23. IMMUNOPROFILE OF PAPILLARY LESIONS

24. Papilloma
with ADH
vs
Papilloma
with DCIS
Both show absent MEC
markers within
Both show present MEC
around the ducts
Positive 34βE12, CK5/6 is
within proliferative
component is indicative of
ADH

25. Intracystic
papillary
carcinoma vs
Solid papillary
carcinoma
Both show absent
MEC around ducts
Intracystic papillary
ca shows positive
staining by Collagen
IV around ducts

26. Case 6
• Papilloma
• Atypical papilloma
• Papilloma with DCIS
• Intracystic papillary
carcinoma
• Solid papillary carcinoma

27. Diagnostic Rationale
• MEC – SMMHC or p63
• Collagen type IV
• Intracystic papillary carcinoma

28. Case 7
ADH vs DCIS
34βE12
ADH

29. Case 8
ADH vs DCIS
34βE12
DCIS

30. DUCTAL
VS
LOBULAR
3

31. Ductal vs Lobular
• This classification is based on cell cohesiveness
• Invasive ductal carcinomas (IDCs) are often unifocal lesions,
compared with invasive lobular carcinomas (ILCs), which are
often multifocal
• Distant metastases from ductal carcinoma preferentially
involve lung and brain, whereas metastases from lobular
carcinoma more often involve the peritoneum, bone, bone
marrow, and visceral organs of the gastrointestinal (GI) and
gynaecologic tracts
• In spite of the above differences, at present, with combined
multimodality therapy, no difference in disease-free or
overall survival is apparent between ductal and lobular
carcinomas.

32. E cadherin
and p120ctn

33. E cadherin
• Strong E-cadherin (ECAD) membranous staining has been long used to
define ductal carcinoma
• Ductal carcinomas, both in situ and invasive, retain membranous
ECAD
• Lobular carcinomas studied at the genetic level have often shown
mutation that accounts for the loss of cohesiveness
• This correlates with either a complete absence of the ECAD protein or
abnormal localization (apical or perinuclear)

34. Case 9
Insitu ca
DCIS? LCIS?
E cadherin

35. ILC
aberrant staining with E cad

36. p120ctn
• When ECAD is absent, the cytoplasmic pool of p120 increases.
• Therefore in normal ducts and in ductal carcinomas, p120 shows a
membranous pattern of staining
• In contrast, lobular carcinomas, with absent or non functional E-
cadherin, show strong cytoplasmic p120 immunoreactivity

37. Case 10
Ecad – brown
p120 - pink
Membranous for
both
Ductal

38. Case 11
P120 cytoplasmic, Invasive and ILC

39. Paget’s disease
4

40. Paget’s disease
• The presence of malignant epithelium with breast cancer
immunophenotype (CK-7+) within the nipple epidermis
• The majority (>90%) of the underlying breast carcinomas are high
grade ductal carcinoma in situ with apocrine differentiation and
comedo-necrosis, they are her 2 neu positive and ER PR negative
• Accordingly the Paget cells are also her 2 neu positive and ER, PR
negative
• The differential for Paget disease includes melanoma and squamous
cell carcinoma in situ aka (Bowen disease)

41. Case 12
CK 7
Her 2

42. Pitfalls – Toker cells
• They are CK 7 positive cells present in skin of normal
nipple, however they are cytologically bland and
usually do not cause diagnostic problems

43. Pitfalls – Pseudo
Paget
• Large histiocytes infiltrate the epithelium
and impart a picture that simulates Paget
disease
• Cells are CK7 negative and are strongly
positive for CD68

44. Detection of
lymphatic space
invasion
5

45. Lymphatic space invasion
• Peritumoral lymphatic space invasion, is a known determinant of
lymph node metastasis
• However the interpretation in paraffin sections is complicated by
• Retraction artefacts
• Ducts with misplaced epithelium
• Artefactual displacement of cells

46. ________ shows high sensitivity and specificity for normal lymphatic endothelium
D2-40
Prominent staining
around lymphatic,
smudgy around
duct

47. Identify the
stain
CD31

48. Sentinel lymph
node invasion
a soldier or guard whose job is to stand and keep watch
6

49. Rationale
• Historically, complete axillary lymph node dissection was performed with
lumpectomy or mastectomy specimen, primarily for staging purposes,
providing information that was used to determine adjuvant chemotherapy
• The morbidity associated with this procedure is substantial in terms of arm
pain, limitation of arm motion, and chronic lymphedema
• The SLN is identified by injecting a radioisotope and blue dye before
planned surgical excision, Identified by a combination of visual inspection
for blue dye and intraoperative scanning for radioactivity
• The rationale for this approach is that for patients who are SLN negative, a
further morbid procedure of axillary cleanout is unnecessary

50. SLN invasion
• SLNs are histologically examined on multiple
levels and by cytokeratin stains (AE1/AE3) on at
least two levels
• If the primary breast carcinoma is of the ductal
type, most pathologists would agree that they
would be able to identify micro metastases (0.2-
2mm) on HE. Therefore cytokeratin stains on
SLN do not add any significant information in a
primary ductal cancer
• However, when the primary breast tumor shows
a lobular morphology; Because of single-cell
infiltration, small metastases (micro metastases)
of lobular carcinoma require IHC

51. Systemic
metastases of
breast carcinoma
7

52. To know whether breast is the primary
• GCDFP-15
• PPV 99%
• Also stains includes acinar structures in salivary glands, apocrine glands, and sweat
glands and in Paget disease of skin, vulva, and prostate
• ER and PR
• Mullerian tumors may be positive
• MAMMAGLOBIN
• Endometrioid carcinomas and melanomas may be positive
• PAX-8
• Useful negative marker for breast carcinomas
• Positive in tumors of Mullerian origin

53. Case 12
60/F, sections from peritoneal
mass
CK - 7 GCDFP-15MAMMAGLOBIN

54. Fibro epithelial
tumors
8

55. Fibroadenoma vs
Phyllode’s
• Phyllode’s expresses CD34 in stromal cells
indicating fibroblastic/myofibroblastic
origin, fibroadenoma does not

56. Benign vs malignant
phyllode’s
• Ki67 labelling indices range
from
• Benign – 1-5%
• Borderline – 6-16%
• Malignant – 12-50%

57. Theranostics
Theranostics is a new
field of medicine which
combines specific
targeted therapy based
on specific targeted
diagnostic tests
9

58. Theranostics
• Pathologic features of breast carcinoma that have prognostic value
include
1. tumor size, grade, and histologic type
2. lymph node status
3. expression of HRs
4. HER2 expression

59. ER/PR
• Recognition that estrogen ablation had an impact on groups of
patients with breast cancer and that clinical responsiveness
correlated with the expression of ER is the rationale behind testing for
ER and PR
• One of the effects of estrogen is to induce the PR (ER and PR + tumors
form 99% of receptor positive tumors) and ER-negative, PR-positive
tumors account for fewer than 1% of all breast cancers
• Nuclear immunostaining for both receptor proteins can be
demonstrated in normal breast acini (15-20%), which serve as
internal controls for the testing procedure

60. ALLRED
SCORE
The degree of ER and PR staining has consistently been shown to identify
groups of patients with significantly different risks of overall survival, time to
recurrence, and treatment response to hormonal therapy.
1 2
3 54
0

61. Her2/neu(ERBB2)
• Approximately 15% to 20% of breast cancers demonstrate HER2 gene
amplification and/or protein overexpression
• In the absence of adjuvant systemic therapy, HER2-positive breast
cancer patients have a worse prognosis, that is, they have higher rates
of recurrence and mortality
• HER2 positivity is predictive of response to anthracycline- and taxane-
based therapies
• It is also important to note that HER2- positive tumors generally show
relative resistance to tamoxifen
• Trastuzumab is the Theranostic treatment for these tumors

62. SUMMARY
1. Combination of SMMHC and p63 is ideal for marking myoepithelial cells
2. Papilloma with ADH vs Papilloma with DCIS - Positive 34βE12, CK5/6 is
within proliferative component is indicative of ADH
3. Intracystic papillary carcinoma vs Solid papillary carcinoma - Intracystic
papillary ca shows positive staining by Collagen IV around ducts
4. Strong E-cadherin (ECAD) membranous staining has been long used to
define ductal carcinoma
5. Lobular carcinomas show strong cytoplasmic p120 immunoreactivity
6. Paget’s disease - The presence of malignant epithelium with breast
cancer immunophenotype (CK-7+) within the nipple epidermis

63. SUMMARY
7. D2-40 shows high sensitivity and specificity for normal lymphatic
endothelium and helps in detection of lymphatic space invasion
8. Sentinel lymph node invasion - SLNs are histologically examined on
multiple levels and by cytokeratin stains (AE1/AE3) on at least two levels
9. GCDFP, ER/PR and mammaglobin are positive markers to detect systemic
metastasis, PAX 8 is a useful negative marker
10. Phyllode’s expresses CD34 in stromal cells, fibroadenoma does not
11. Ki67 labelling indices help in differentiating benign, borderline and
malignant phyllode’s
12. ER/PR positivity – tamoxifen, HER2 positivity - Trastuzumab

64. THANK YOU