Molecular pathogenesis and tests in Ca breast

1. MOLECULAR TESTING IN
CARCINOMA BREAST
Dr. Aysha Byju Rahim
Final year PG resident

2. Objectives
To give a brief overview of the molecular
pathogenesis of breast cancer
Classification systems in breast cancer – intrinsic
subtypes
To provide an update on molecular testing in current
clinical practice
Prognostic and therapeutic values of each test and
the methodology used.

3. Synopsis
Introduction
Classification of breast cancer into molecular subtypes
Tests done in routine practice –Basis, Methodology,
clinical applications
 Hormone receptors (ER,PR), HER2 testing,Ki67
proliferative index (IHC4 Assay)
MAAAs(Multianalyte assays with algorithmic analysis)
Germline testing in breast cancer
Conclusion

4. INTRODUCTION
■ Emerging molecular classifications of breast cancers - novel
perspectives to the assessment of individual cases, and
opportunities for better treatments.
■ Shed light on distinct biological subsets of triple-negative breast
cancers, for which new targeted therapies are being developed
■ Very common, heterogenous group ; with intratumour and
intertumour heterogeneity
– major challenge to the success of therapy
■ Distinct biological behavior

5. • Following introduction of
mammographic screening
in the 1980s- the number
of cases of DCIS and
invasive carcinoma
increased , older women.
• The number of women
screened - recently
plateaued, as has the
incidence of breast cancer
Epidemiology

7. Molecular Mechanisms of Carcinogenesis
and Tumor Progression

8. Driver mutations
■ proto-oncogenes PIK3CA,
■ HER2
■ MYC
■ CCND1 (which encodes cyclin D1),
■ the tumor suppressor genes TP53
■ (in familial cancers) BRCA1 and BRCA2

9. Molecular and immunophenotypic
classification of breast carcinoma
■ Perou et al and Sorlie et al
■ Classified breast carcinoma 4 intrinsic subtypes: luminal A,
luminal B, HER2/neu enriched, and basal-like
■ Was defined based on a relatively small number of cases,
having missed out the less common distinctive types of
breast cancer (such as secretory carcinoma)

10. INTRINSIC SUBTYPES AND THEIR
SURROGATES
■ Rare intrinsic subtypes
– claudin-low, with low expression of cell adhesion genes
– Apocrine-like, which often expresses androgen receptors
■ Developed with PAM 50 risk of recurrence score

11. Test Methodology used
■ Subtyping by gene expression profiling
■ Prosigna assay (NanoString Technologies, Seattle,
Washington)
■ HR and HER2 status can be used as a surrogate for
intrinsic subtyping by molecular methods

12. ■ ER-positive, HER 2- negative ( “luminal,”50% to 65% of
cancers) : most common form of invasive breast cancer
– ER-positive, HER2-negative, low proliferation (40% to
55% of cancers): majority of cancers in older women
and in men, lowest incidence of local recurrence and
cured by surgery
– ER-positive, HER2-negative, high proliferation (approx.
10% of cancers): most common type of carcinoma
associated with BRCA2 germline mutations
INTRINSIC SUBTYPES

13. ■ HER 2-positive (approximately 20% of cancers) : second
most common molecular subtype of invasive breast cancer
– Excellent prognosis
■ ER-negative, HER 2-negative tumors (“basal-like” triple
negative carcinoma; approx. 15% of cancers): third major
molecular subtype, more common in young
premenopausal women, BRCA 1 mutation carriers
INTRINSIC SUBTYPES

15. LUMINAL A SUBTYPE
ER/PR +
HER 2 -
Ki67 INDEX- LOW
LUMINAL B SUBTYPE
ER/PR +/-
HER 2 –
Ki67 INDEX- HIGH
HER 2 ENRICHED
ER-
PR-
HER2 +
BASAL LIKE
ER –
PR –
HER 2 -
Lobular Invasive Ca NST
High grade
50% of apocrine
carcinomas
Invasive Ca NST
High grade
Papillary Micropapillary
carcinoma
40% of micropapillary
carcinomas
Carcinomas with
medullary features
Cribriform Metaplastic
carcinoma
Mucinous Adenoid cystic
Tubular Secretory
Invasive Ca NST low
grade

18. Claudin low subtype
■ Recent studies revealed - claudin-low subtype
■ characterized by down-regulation of tight junction proteins
including E-cadherin, occludin, and some claudins
■ by high expression of genes associated with epithelial-
mesenchymal transition (EMT), immune response, and the
breast cancer stem-cell phenotype
■ generally triple negative
■ metaplastic carcinomas

20. MOLECULAR TESTS
Basis of each test
Test methodology
Clinical Applications

22. 1.HORMONE RECEPTOR TESTING(ER & PR)
■ BASIS OF THE TEST : ancillary testing supports diagnosis,
classification, prognosis, and prediction of response to
therapy
■ Hormone receptor status - determined by the tumor
cells’ expression of nuclear receptors for ER & PR
■ Inhibiting the stimulatory effect of steroid hormones :
important component of therapy .

23. Test Methodology
■ Biochemical ligand-binding assays - initially used;
cumbersome, could not be performed on routine FFPE
■ IHC based assays replaced them
■ Allred scoring method: the proportion (on a scale from 0 to
5) and intensity (from 0 to 3) of staining are summed to give
an overall score.

26. Sensitivity of Allred method = 99.4%
Specificity of Allred method = 99.5%
Sensitivity of conventional score= 88 %
Specificity of conventional score= 84%.
Allred scoring for ER reporting and it's impact in clearly distinguishing ER negative from ER positive breast cancers
Asim Qureshi, Shahid Pervez
Department of Pathology, Shaukat Khanum Cancer Hospital, Lahore
Department of Histopathology, Aga Khan University Hospital, Karachi

27. Clinical Implications
■ predicts response to endocrine therapy including tamoxifen
and aromatase inhibitors.
■ National Comprehensive Cancer Network (NCCN) guide-
lines recommend endocrine therapy for any patient with
HR+ breast cancer.
■ All new and recurrent breast cancers must be tested for ER
and PR expression.

28. 2. ERBB2 (HER2) TESTING
■ Basis of Test
■ The erb-b2 receptor tyrosine kinase 2 (ERBB2, also known
as HER2) gene : overexpressed in 20% to 30% of breast
cancer
■ tyrosine kinase receptor
■ expressed at the cell surface
■ homodimeric form, as well as in heterodimers with other
ErbB family members

29. Test Methodology
■ ASCO and CAP, 2007 consensus statement  updated in
2011 and 2013: provide guidance on HER2 testing in breast
cancer.
■ Key aspects
– All primary, recurrent, and metastatic breast cancers be
tested for HER2, either by IHC( to detect overexpression)
or FISH( to detect amplification)
– Both IHC and FISH have the potential to return a positive,
negative, or equivocal result.
– Equivocal cases are recommended to undergo reflex
testing by the other modality

30. 2013 HER2/neu scoring criteria by IHC
Score 0: No staining observed or membrane staining that is incomplete
and faint/barely perceptible and within 10% or less of the invasive
tumor cells: negative for HER2/neu overexpression.
Score 1+: Incomplete membrane staining that is faint/ barely
perceptible and within more than 10% of invasive tumor cell: negative
for HER2/neu overexpression
Score 2+: Circumferential membrane staining , incomplete and/or
weak/moderate and within more than 10% of the invasive tumor cells,
or complete and circumferential membrane staining that is intense and
within 10% or less of the invasive tumor cells: equivocal
requires reflex testing by in situ hybridization for HER2/neu
amplification.
Score 3+: Complete intense circumferential membrane staining in more
than 10% of the invasive tumor cells: Positive for HER2/neu
overexpression

32. Three important points
(2013 HER2/neu scoring criteria )
(1)To render a 3+ score, completely circumferential and
very strong membranous staining is required
(2)The definition for HER2/neu IHC 2+ will be ‘‘weak to
moderate complete membrane staining that is
observed in more than 10% of tumor cells”
(3)‘‘If the initial HER2 test in a core needle biopsy of a
primary breast cancer is negative, a new HER2 test
must be ordered on the excision specimen”

33. HER2 testing by FISH
■ uses probes directed at the HER2 gene
■ generally reported as a ratio normalized to
chromosome 17 centromeric probes (HER2:CEP17
ratio).A ratio less than 2.0 with average HER2 copy number of fewer than 4.0
signals per cell - no HER2 amplification
A ratio of 2.0 or greater regardless of HER2 copy number, or a ratio of less
than 2.0 with 6.0 or more HER2 signals per cell, - amplification
Cases with a ratio less than 2.0 and with 4.0 to 6.0 signals per cell-equivocal

36. Clinical Implications
■ HER2 amplification -independent risk factor for recurrence
and death
■ Trastuzumab, a humanized monoclonal antibody against
HER2; improves progression-free survival and overall survival,
for both early high-risk disease and metastatic disease.
■ Pertuzumab, a second-generation monoclonal antibody, and
lapatinib, a small-molecule kinase inhibitor: FDA approved
■ Triple-negative tumors - less favorable prognosis

37. 3.Ki-67 PROLIFERATION INDEX
■ The Ki-67 antigen : expressed in the nuclei of cells that are in
cycle (ie, not in G0 phase) , reflects cell proliferation
■ measured by immunohistochemistry: analytic validity has
not been well established,formal inter-laboratory
standardization is not in place.
■ St Galen International breast conference,2013 recommends
inclusion of Ki67 index ≥20% in defining Luminal B subtype

38. 4. NEXT-GENERATION SEQUENCING
■ BASIS OF TEST : sequence large numbers of genes to
identify actionable mutations in the tumor
■ Clinically actionable mutations are those that are either
prognostic or predictive
■ Helps identify “driver mutations” from passenger
mutations

39. Test Methodology
■ ‘‘gene panel testing’’
■ Next-generation sequencing detect: single-
nucleotide variants, insertions/deletions, copy-
number variants, and structural variants
(rearrangements, translocations).
■ both cost- and time-effective compared with single-
gene testing.

40. Clinical Implications
■ Provides information on the genomic landscape of breast
cancer
■ triple-negative tumors: greater genetical heterogeneity
■ Only 7 out of 40 cancer genes( 58%) assosciated with
breast cancer recognised by TCGA network : Actual driver
mutations
■ TP53, PIK3CA,MYC,ERBB2,FGFR1,CCND1 and GATA3

41. Novel therapeutic agents
a)poly(ADP-ribose) polymerase (PARP) inhibitors for
breast tumors with BRCA mutations
b) Triple-negative breast cancers with amplification of the
fibroblast growth factor receptor genes FGFR1 or FGFR2.
- FGFR inhibition has been investigated,

42. MULTIANALYTE ASSAYS WITH ALGORITHMIC
ANALYSIS
■ Basis of Test: in vitro diagnostic test
– combines measurements of multiple genes or other
analytes to derive predictive or prognostic
information
■ The measurements are integrated using a specific, closed-
form, often proprietary algorithm to yield a clinically validated
result
■ based on measuring mRNA levels for selected genes.

43. ■ MammaPrint (Agendia, Amsterdam, the Netherlands,
and Irvine, California)
■ Oncotype DX (Genomic Health, Redwood City,
California)
■ Prosigna Assay(Nanostring technologies, Seattle, WA
,USA)

44. MAMMAPRINT : Test Methodology
■ measures the expression of 70 genes
■ using a microarray platform
■ reports a binary result (low risk or high risk) for
recurrence without adjuvant chemotherapy at 10 years
■ On Fresh or FFPE also

45. Clinical Implications
■ The assay has now been validated in both lymph
node– positive and lymph node–negative patients,
and in both ER+ and ER- tumors
■ Estimates risk of recurrence
■ intended to spare patients at low risk of recurrence
from receiving adjuvant chemotherapy, with its
attendant morbidity
■ not intended to predict the response, per se, to
chemotherapy

46. ONCOTYPE Dx BREAST CANCER ASSAY
■ Basis of Test: based on reverse transcriptase–polymerase
chain reaction (RT PCR) analysis of 21 genes.
■ Initially validated for ER+, HER2-, node-negative invasive
cancer
■ the validation has now been extended to node-positive disease
(1–3 nodes) as well

47. Test Methodology
■ The 16 cancer-related genes included in the final assay fall
into several groups:
– a proliferation group, an ER-related group, an invasion-
related group, and several miscellaneous genes. Five
housekeeping genes are used for normalization.
■ A formula is used to calculate a recurrence score between 0
and 100, which is further trichotomized into low, medium,
or high risk.

48. Clinical Implications
■ predict risk of recurrence at 10 years in node-negative
patients,
■ predict the benefit of adding chemotherapy with
cyclophosphamide, doxorubicin, and fluorouracil—or
cyclophosphamide, methotrexate, and fluorouracil—to
tamoxifen
■ predict recurrence in patients who have had a local excision
(partial mastectomy) for ductal carcinoma in situ.

49. PROSIGNA BREAST CANCER ASSAY
■ Basis of Test : based on tumor gene expression, but differs in
that it represents a clinical implementation of the intrinsic
subtype concept
■ design a classifier able to reproducibly identify the intrinsic
subtype of a tumor based on the expression of a smaller set
of genes
■ Prediction Analysis of Microarray-50 (PAM50) classifier

50. Test Methodology
■ fifty PAM 50 genes and 8 housekeeping genes
■ measured on RNA extracted from formalin-fixed, paraffin-
embedded tissue on the NanoString Counter Analysis
System.

51. Clinical Implications
■ provides prognostic information and predicts the
effectiveness of chemotherapy.
■ facilitating treatment decisions
■ not performed in a single, centralized laboratory,instead is
performed in multiple reference laboratories,using the
same platform, probe library, and algorithms
■ have benefits from the standpoint of turnaround time,
proficiency testing, and other regulatory concerns

52. GERMLINE TESTING IN
BREAST CANCERS

53. Familial Breast Cancer
■ Approximately 12% of breast cancers occur due to
inheritance of an identifiable susceptibility gene or genes
■ multiple affected first-degree relatives, early onset
cancers, multiple cancers, or family members with other
specific cancers
■ Mutations in BRCA1 and BRCA2 are responsible for 80%
to 90% of “single gene” familial breast cancers and about
3% of all breast cancers

55. 7. GERM LINE TESTING IN BREAST CANCER
■ Basis of Test :
■ BRCA1 or BRCA2: account for approximately 90%
■ BRCA1 (on chromosome 17q21) and BRCA2 (on
chromosome 13q12.3)

56. Other inherited syndromes
■ Li-Fraumeni syndrome (TP53 mutation)
■ Cowden syndrome (PTEN mutation)
■ Peutz Jeghers syndrome (serine/threonine kinase 11
[STK11] mutation)
■ hereditary diffuse gastric cancer (cadherin 1 [CDH1]
mutation)
■ ataxia-telangiectasia (ATM serine/ threonine kinase
[ATM] mutation).

57. Test Methodology
■ DNA sequencing from non tumor tissue, often peripheral
blood
– Sanger sequencing: covering individual genomic
regions of up to approximately 1 kb
– NGS: covering multiple regions on a single platform

58. Clinical Implications
■ Identify genetic predisposition
■ Counsel patients and relatives
■ Guide screening and treatment

60. CONCLUSIONS
■ Ancillary workup of invasive breast cancer includes non
molecular testing as well as tests based on the
assessment of nucleic acids
■ Non molecular testing includes HRs and HER2 status,
which can also be assessed at the DNA level via FISH
■ Optimal breast cancer care requires that both
pathologists and clinicians maintain a working
knowledge of all of these rapidly evolving techniques.

61. References
■ Recent Advances in Histopathology, 24th edition, Adrienne M Flagnanan

62. ■ Arch Pathol Lab Med. 2016;140:815–824
■ Accepted for publication April 11, 2016. From
the Department of Pathology and Immunology,
Washington University School of Medicine, St
Louis, Missouri. Presented at the 2nd Princeton
Integrated Pathology Symposium: Breast
Pathology; February 8, 2015; Plainsboro, New
Jersey.

63. THANK YOU