This document discusses the molecular classification of breast cancer and its role in patient management. It begins by explaining the need for molecular classification in diagnosis, prognosis, prediction, and management of breast cancer patients. It then covers various molecular alterations and genetic factors that contribute to breast cancer development, including growth factor receptors, signaling molecules, cell cycle regulators, and adhesion molecules. The major subtypes of breast cancer - luminal A/B, HER2-enriched, and basal-like - are identified based on expression of estrogen receptor, progesterone receptor, HER2, and Ki-67. Molecular classification is important for determining prognosis and guiding treatment decisions.

1. Molecular classification of breast carcinomas
and its role in patient management

2. Why do we need a mol classification?
1 Diagnosis
2 Prognosis
3 Prediction
4 Management

3. INTRODUCTION
• Breast cancer is a heterogenous disease caused by
interaction of both inherited and environmental risk factors.
• There is progressive accumulation of genetic and epigenetic
changes in breast cancer cells.

4. INTRODUCTION
• Tumors with similar clinical and pathological presentations
may have different behaviours.
• Better understanding of the molecular classes of breast
cancer, may also lead to new biological insights and
eventually to better therapies that are directed toward
particular molecular subsets.

5. GENETICS

6. GENETICS – Breast cancer predisposition genes
Low penetrance, high
frequency
Mod penetrance, low
frequency
High penetrance, low
frequency
FGFR CHEK2 BRCA1
LSP1 BRIP1 BRCA2
TOX3 ATM TP53
PALB2 PTEN
CDH 1
STK 11

7. GENETICS – Breast cancer predisposition genes
BRCA -1 ( Chr.17) BRCA-2 (Chr.13) Tp53( Chr.17) CHEK2( Chr. 22)
FUNTIONS: 1. Transcription
2. DNA Repair of
double stranded
breaks
3. Ubiquitination
4. Transcriptional
regulation.
1. Stability of the
human genome
2. DNA double
strand break
repair.
1. Cell cycle control
2. DNA replication
3. DNA repair
4. Apoptosis.
1. Cell cycle
checkpoint kinase,
recognition and
repair of DNA
damage.
2. Activates BRCA1
and p53 by
phosphorylation
Germline point
mutations/Deletions
Mutations- 20% Mutations
- Sporadic breast
cancers.
- Li fraumeni syndrome
Mutations - <5%
Li fraumeni variant
Increase breast cancer
risk after radiation
exposure
Hereditary breast &
ovarian cancers.
Hereditary breast
cancer, ovarian cancer,
increased cancer risk in
male carriers

8. Molecular alterations
• (1) Growth receptor overexpression (HER2/neu amplification,
EGFR, FGFR1 or FGFR2).
• (2) Growth factor overexpression (FGF1/FGF4).
• (3) Intracellular signaling molecule alterations (HRAS mutation)
• cell cycle regulator alterations
(TP53 mutation, RB inactivation, CCND1 gene amplification);
• adhesion molecule alterations (⬇E-cadherin, ⬇P-cadherin,
⬆cathepsin); and
• others (CMYC amplification).

9. Pathogenesis – Hormonal factors
• Breast growth increases during puberty,
menstrual cycles, and pregnancy under
the influence of hormones. This also
includes the risk of proliferation of cells
with DNA damage.
• If there is premalignant or malignant
cells, hormones stimulate their growth as
well as the growth of normal epithelial
and stromal cells- “ TUMOR
DEVELOPMENT”.
• Metabolites of estrogen will generate
DNA – damaging free radicals.

11. Classification of breast cancer
• The existing histological classifications do not fully capture the
varied clinical course of this disease.
• Histological type,
• Grade
• Tumor size
• Lymph node involvement, and
• Estrogen receptor α (ER) and HER-2 receptor status all
influence prognosis and the probability of response to
systemic therapies.

13. Histological classification of breast cancer

14. Changes in recent WHO Classification of
Tumors of the Breast.
1)Carcinoma with medullary features
• Medullary carcinoma
• Atypical medullary carcinoma
• Invasive carcinoma NST with medullary
features
2) Signet ring cell carcinoma is a now a
separate entity under special type.
3) Carcinoma with neuroendocrine features
• Neuroendocrine tumor, well
differentiated
• Neuroendocrine carcinoma, poorly
differentiated (small cell carcinoma)
• Carcinoma with neuroendocrine
differentiation
4) Metaplastic carcinoma of no special type
• Low-grade adenosquamous carcinoma
• Fibromatosis like metaplastic carcinoma
• Squamous cells carcinoma
• Spindle cell carcinoma
• Metaplastic carcinoma with
mesenchymal differentiation
• Chondroid differentiation
• Osseous differentiation
• Other types of mesenchymal
differentiation
• Mixed metaplastic carcinoma
• Myoepithelial carcinoma

15. Changes in recent WHO Classification of Tumors of
the Breast (cont..)
5) Mucoepidermoid tumor is now included
in rare type tumors ( earlier it was
classified under metaplastic carcinoma)
6) Precursor lesions
• Ductal carcinoma in situ ( earlier it was
classified under intraductal proliferative
lesion)
• Lobular neoplasia
• Lobular carcinoma in situ
• Classic lobular carcinoma in situ
• Pleomorphic lobular carcinoma in
situ
• Atypical lobular hyperplasia
7) Intraductal proliferative lesions
• Usual ductal hyperplasia
• Columnar cell lesions including flat
epithelial atypia
• Atypical ductal hyperplasia
6) Papillary lesions
• Intraductal papilloma
• Intraductal papilloma with atypical
hyperplasia
• Intraductal papilloma with ductal
carcinoma in sit
• Intraductal papilloma with lobular
carcinoma in situ
• Intraductal papillary carcinoma
• Encapsulated papillary carcinoma
• Encapsulated papillary carcinoma with
invasion
• Solid papillary carcinoma
• In situ
• Invasive

16. Changes in recent WHO Classification of Tumors of
the Breast (cont..)
• Epithelial-myoepithelial tumors
• Pleomorphic adenoma
• Adenomyoepithelioma
• Adenomyoepithelioma with carcinoma
• Adenoid cystic carcinoma
• Malignant lymphoma
• Diffuse large B cell lymphoma
• Burkitt lymphoma
• T cell lymphoma
• Anaplastic large cell lymphoma, ALK negative
• Extranodal marginal-zone B cell lymphoma of MALT-type
• Follicular lymphoma

17. Invasive carcinoma, No Special Type (NST)
• Histologic Grading is based on tubule formation, nuclear
pleomorphism and mitotic rate.
• Nottingham histologic score system- scarff-Bloom-Richardson
grading system:
Parameters 1 2 3
Tubule formation >75% 10-75% <10%
Nuclear
pleomorphism
Uniform Mild variation Marked
variation
Mitosis /10 hpfs 0-5 6-10 >10
Grade 1 3,4 and 5
Grade 2 6 and 7
Grade 3 8 and 9

18. Molecular classfication
• It is generally accepted that the different clinical courses of
patients with histologically identical tumors is a result of
molecular differences among cancers.
• The routine immunohistochemical (IHC) analysis for ER, PR,
and HER2 provides critical prognostic and predictive
information for Invasive breast cancers.

19. The subtypes of breast cancers include
• Luminal (type A and B, and questionable type C)
luminal A – ER +ve, PR +/-, HER2 –ve
luminal B / HER2 Negative like - ER +ve, PR +/-, HER2 –ve , high
Ki67 index
luminal B / HER2 positive like - ER +ve, PR+/-, HER2 +ve, high
Ki-67 index (triple positive)
• HER2/neutype,
• Basal-like, and
• Normal breast-like.
• The last subtype is most likely an artifact rather than a genuine type of
breast cancer, resulting from lack or paucity of tumor in the tissue
sample used for the microarray analysis.

20. According to the St. Gallen International Expert Consensus recommendations 2011, five
molecular subtypes of invasive breast cancer have been differentiated by their expression of the
IHC markers ER, PR, HER2, and Ki-67:
Intrinsic suptype ER and/or PR HER2 Ki-67
Luminal A-like (LumA) + − <14 %
Luminal B/HER2
negative-like
(LumB/HER2 neg.) + − ≥14 %
Luminal B/HER2
positive-like
(LumB/HER2 pos.) + + any
HER2-type (HER2) both− + any
Triple negative (TN) both− − any

21. Major pathways of breast cancer development

22. Molecular classification of breast tumors
Diagnostic modalities
• Immunohistochemistry (IHC) was developed more than 25 years
ago and currently forms the cornerstone of molecular classification
of breast cancer into ER-positive and ER-negative categories.
• Nucleic acid in situ hybridization was introduced more than 15
years ago and is now also routinely used to classify breast cancer
into HER-2 amplified or nonamplified categories.
• More recently, high throughput proteomic and gene-expression
profiling methods are being explored as diagnostic tools.

23. Estrogen receptor (ER)
• ER is a nuclear protein with 1 DNA-binding domain and 2 activation
function domains.
• 65% to 75% of IBCs express ER, with ER being a major contributor
to its development.
• Clinically, ER +ve IBCs are usually better differentiated - favorable
prognosis - respond to hormonal therapy.

24. Estrogen receptor (ER)
• The American Society of Clinical Oncology/College of American
Pathologists (ASCO/CAP) guideline recommends that ER be
considered positive if 1% or more of tumor cells have nuclear
staining of any intensity.
• Allred scoring and H-scoring are two other commonly used systems
for ER and PR evaluations

25. Progesterone receptor (PR)
• Similar to ER, PR is also a transcription factor, largely controlled by ER
and to some degree by growth factors as well, and is expressed in 55% to
65% of IBC.
• In most cases, PR coexpresses with ER.
• Clinically, loss of PR suggests a more aggressive behavior and less
response to hormonal therapy.
• The ASCO/CAP guideline recommends that PR be considered positive if
1% or more of tumor cells have nuclear staining of any intensity.

26. ASCO/CAP guideline for IHC testing of ER/PR
• Tumors having 1% or higher invasive cancer cells staining - +ve
• The average intensity of stain must be included (weak, moderate,
strong).
• Interpret weather the sample is positive or negative.
• Use of composite score based on percentage plus intensity is
optional (Allred, H, Quick scores)
• Specimens placed in 10% neutral buffer formalin no later than 1hr.
• Fixation time atleast 6hrs and no longer than 72hrs.
• Normal breast cells in the sample – positive control.

27. Modified McCarty’s H-scoring system/ Histoscore:
score Interprestation
O-50 Negative
50-100 Weakly positive
101-200 Moderately positive
201-300 Strongly positive

28. Human epidermal growth factor receptor
(HER2)
• HER2 encodes a transmembrane tyrosine kinase receptor that binds
to its extracellular signals and initiates a signaling cascade mediating
cell proliferation and survival.
• About 12% to 20% of IBCs either overexpress the HER2 protein -
aggressive tumor growth and poor clinical outcome.
• Its expression is also indicative of a potential response to anti-HER2
therapy.

29. Human epidermal growth factor receptor
(HER2)
• The ASCO/CAP guideline recommends that HER2 be defined as
positive if 10% or more of tumor cells exhibit strong uniform
membrane staining.

31. ASCO/CAP guideline for IHC testing of
HER2/neu testing
Positive HER2/neu result :
• An IHC staining of 3+ (uniform, intense membrane staining of >30% of
invasive tumor cells) or
• FISH result of >6 HER2/neu gene copies or
• FISH ratio of >2.2. (HER2/neu signal: chromosome 17 signal).
Negative HER2/neu result:
• IHC staining of 0/1+ or
• FISH result of <4 HER2/neu gene copies per nucleus or
• FISH ratio of <1.8.
• [ If 1+/2+ is obtained then FISH is preformed]

32. Ki-67
• Ki-67 is a nuclear marker for proliferation expressed in all phases of the cell
cycle except G0.
• Clinically, higher Ki-67 expression- higher grade and more aggressive behavior.
• Nuclear staining of any intensity is evaluated.
• Currently, there is no standardized cutoff value for Ki-67.
• However, the threshold designated as high Ki-67 labeling ranged from 3.5% to
35%.

33. Ki-67
• A 13.25% of Ki-67 labeling was proposed as the cutoff to separate
luminal B tumors from luminal A tumors based on PAM50-defined
subtyping.
• Although a 14% cutoff was endorsed in St Gallen in 2011,most of the
panel voted for a cutoff of greater than 20% as the new cutoff in the
2013 St Gallen conference.
• Also, because there is great variability within any given tumor for Ki-
67 labeling, the actual score largely depends on the areas sampled and
analyzed.

34. CK5
• CK5 is a high–molecular weight cytokeratin and is expressed in
normal myoepithelial cells.
• CK5/6 is often expressed in BRCA1-related breast cancers.
• Nielsen et al report that a panel of 4 markers (ER, HER2, CK5/6, and
EGFR) accurately identifies BLBC.
• The cutoffs for its positivity in the literature range from any positive
cytoplasmic staining to 20% of tumor cells.

35. EGFR
• EGFR, also known as HER1, is a member of the HER family.
• Although the IHC expression of EGFR is not indicative of eligibility for
current EGFR-targeted therapy, it has enhanced the identification of
BLBC significantly because of its higher expression and easier scoring
than that of CK5.

36. p53
• p53 is a vital regulator of genomic stability- controlling the cell cycle
and inducing apoptosis when cell damage is beyond repair.
• However, a missense mutation of p53 results in protein stabilization
and accumulation in the nucleus.
• Expression of p53 has been shown to be associated with poor
outcome for patients with IBC in some studies
• Currently, most investigators use 10% of nuclear staining in tumor
cells as the cutoff for p53 positivity

37. Molecular classification of breast tumors:

39. Luminal (ER +ve, HER2/neu –ve)
• 50 – 65% of cancers
• Most common invasive breast cancer
• It is further divided into two sub groups:
1) Luminal A
2) Luminal B.

40. Luminal A (ER/PR +ve, HER2 –ve, Ki-67 <14%)
• 40 – 55%
• Older women and men
• Detected in mamographic screening and menopausal hormone therapy.
• Diagnosed at an early stage, low incidence of local recurrrence, cured by
surgery.
• High expression of luminal cytokeratin, ER/PR, and absent HER2
• Well to moderately differntiated lobular, mucinous, tubular carcinomas,
cribriform and low grade ductal carcinoma NST.
• Respond well to hormonal therapy (tamoxifen), and incomplete response
to chemotherapy.
• Good prognosis.

41. Lumianl B (ER +ve, HER2 +/–ve, Ki-67 >14%)
• 10%
• ER +ve but ER levels are low and PR +/- (mod to weak expression)
• Variable HER2 expression.
• Higher proliferation (>14%)
• Associated with BRCA2 germline mutations
• mRNA pattern is similar to ER+ve tumors but there is high proliferation
• 10%show complete response to chemotherapy- better prognosis.
• Poorly differentiated lobular carcinoma,IDC-NST, Micropapillary
carcinoma

42. HER2- POSITIVE
• 20%
• Young women, non-white women
• Germline TP53 mutations.
• Complex interchromosomal translocations, high level amplifications of
HER2, high mutational load.
• High expression of HER2, low ER+
• High grade IDC-NST
• Respond to trastuzumab (herceptin), and anthracyclin based chemo
• Poor prognosis
• Metastasize when small in size and early in course, often to viscera and
brain.

43. HER2- POSITIVE
• The introduction of transtuzumab (Herceptin) binds specifically and
inhibits HER2 activity.
• Not all HER2 overexpressing tumors respond, some do become
resistant to treatment.
• Some express truncated forms of HER2, that lacks the transtuzumab
binding site.

44. HER2- POSITIVE
• Numerous therapautic interventions are under trial to overcome
resistant cases.
• Newer antibodies that bind to different HER2 epitopes.
• Dual tyrosine kinase inhibitors that target both EGFR and HER2.
• Antibody toxin conjugates.
• Inhibitors of PI-3 kinase and AKT.

45. ER –ve, HER2 –ve (Basal like)
• 15%
• Young premenopausal and african american, hispanic women.
• Associated with BRCA1 & Tp53 mutations.
• Hihj expression of basal cytokeratins and low exprssion of
HER2/ER/PR.
• High grade IDC-NST, metaplastic carcinoma, medullary carcinoma.
• No respone to hormonal therapy, sesitive to platinum based chemo
and PARP.
• Some cases features overlap with other molecular subtypes.
• Thus gene amplification studies must be done to target specific
treatment.

46. Use of immunohistochemistry as surrogate marker for the
molecular subtypes of breast cancer
MOLECULAR SUBTYPE
Immunoprof
ile
Luminal A Luminal B HER2/neu Basal-like
ER, PR
ER and/or
PR+
ER and/or
PR+
ER–, PR– ER–, PR–
HER2 and
others
HER2–
Low Ki-67
(<14%)
HER2+ or
HER2–
Ki-67 ≥14%
HER2+
HER2–
CK5/6 and/or
EGFR+
EGFR, epidermal growth factor receptor.

48. Histopathology of few tumors

49. Invasive carcinoma, No Special Type (NST)
Majority of tumors (70% - 80%)
Gross: Firm to hard, irregular border, +/-
foci of calcification.
Microscopy : diffuse sheets, well-defined
nests, cords, or as individual cells.
The tumor cells vary in size and shape,
prominent nucleoli, and mitotic figures
are more numerous with areas of
necrosis.

50. Invasive lobular carcinoma
Gross : hard, irregular mass, diffuse infiltrative
pattern.
Microscopy: Discohesive infiltrating tumor cells,
India-file like arrangement.
Often include signet-ring cells containing
intracytoplasmic mucin.
• Well-differentiated and moderately
differentiated carcinomas – ER/PR +
• Poorly differentiated tumors – may overexpress
HER2/neu
Pattern of metastasis is different from other breast
cancers – peritoneum, retroperitoneum,
leptomeninges (carcinoma meningitis), GIT,
Ovaries.

51. Medullary carcinoma
Gross : well circumscribed tumor, soft
and fleshy (medulla = marrow).
Microscopy :
solid, syncytium
like sheets of large
pleomorphic cells.
Moderate to
marked
lymphoplasmacyti
c infiltrate
surrounding and
within the tumor.
Pushing border
Poorly-differentiated tumors : ER/PR –ve
HER2 –ve

52. Mucinous/colloid carcinoma
Gross : soft/rubbery, pale grey
blue, gelatinous
Pushing/ circumscibed borders
Microscopy : Arranged in clusters and small
islands with large lake of mucin.
Well to moderately differentiated tumors are ER
+ve.

53. Invasive papillary and micropapillary
carcinoma
Rare tumors
Inavasive papillary carcinoma:
ER +ve tumors has favorable
prognosis
Invasive micropapillary
carcinomas: are ER -ve, HER2
+ve and have poor prognosis.

54. Metaplastic carcinoma
• Rare type
• Matrix producing carcinomas,
squamous cell carcinomas, and
carcinomas with prominent
spindle cell component
• ER-PR-HER2/neu –ve
• Poor prognosis

55. Tubular carcinoma
• Well formed tubules, apocrine
snouts are typical, calcifications
within the lumen.
• >95% are ER +ve, HER2/neu -ve

56. Therapy
• Surgery
• Radiation therapy
• Hormonal therapy
• Chemotherapy
• Target therapy

57. Therapy - surgery
• Breast conserving surgeries – evaluation of surgical margins is
important.
• Negative margin – recurrence is related to the vicinity and amount of
carcinoma near the margins.

58. Therapy – chemotherapy
• Significant in metastatic carcinoma
• Curative for patients with positive axillary nodes
• Used as adjunct following local treatment.

59. Therapy – Hormonal
• Tamoxifen – for all stages of ER +ve tumors
• Usually combined with irradiation, +/- adjuvant chemotherapy
• Trastuzumab – is a form of target therapy for HER2+ve tumors
• PRAP (poly(adenosine diphosphate-ribose)polymerase) inhibitor –
associated with BRCA1/2 mutations and basal like carcinomas.

60. Effects of therapy on tumor and normal breast
tissue
Vacuolization of tumor cells
Therapy Tumor cells Normal/non-neoplastis
breast tissue
Radiation Bizzare nucleus, tumor
necrosis
atypia of epithelial cells,
lobular sclerosis and atrophy
Hormonal prominent stromal
fibrosis and hyalinization,
degenerative changes
Chemotherapy morphologic changes –
histiocyte like change
atrophy and occasional
atypia

61. Prognostic factors
1. Patient’s age
2. BRCA1 status
3. Pregnancy and ocps
4. Early diagnosis
5. Invasion +/-
6. Size of the tumor
7. Cytoarchitectural type
8. Microscopic grade
9. Tumor margins
10. Tumor necrosis
11. Microvessel density
12. Mucin
13. HER2/neu
14. p53
15. Skin invasion
16. Nipple invasion
17. Axillary lymph node metastasis

62. Prognostic factors
• Gene expression profiling.
• There are many studies reporting the use of microarray analysis to
select gene signatures for separating prognostic/predictive groups, and
thus will potentially help with selection of therapy.
• Two popular commercially available tests are:
(1) MammaPrint (70 gene expression analysis by microarray, but fresh
or frozen tumor tissue is required); and
(2) Oncotype DX (analysis of expression of 16 cancer-related genes
and 5 reference genes by RT-qPCR, and paraffin-embedded tissues are
used).

63. Summary