The presentation includes recent molecular techniques employed in establishing prognosis and management of breast cancer

1. Molecular Diagnosis in
breast cancer
Moderated By : Dr Moorat S Yadav
Presented By : Dr Ankit Lalchandani

2. • Molecular pathology techniques used commonly in hematological
malignancies and soft tissue sarcomas
• Paradigm shift in classification : Histopathological molecular
• Among epithelial malignancies breast cancer is most extensively
studied
• Led to the concept that breast cancer is a heterogenous disease

3. Molecular classification
• Research by Stanford group classified breast cancer into four subtypes
• Luminal A
• Luminal B
• HER 2 enriched
• Basal like
• Different biological and clinical behaviour and response to therapy

4. • ER positive group :
• Luminal A( ER/PR +ve, Ki67 <14%)
•
• Luminal B ( ER/PR +ve, Ki67 >14%)
• Prognosis of luminal B worse than luminal A

5. • ER negative group :
• Her 2 enriched group : high level of her2
• Basal like : ER/PR/Her -ve, express basal cytokeratins
• Claudin –low tumors : epithelial to mesenchymal transition
• Molecular apocrine subgroup : increased androgen signalling and molecular
apocrine gene profile

6. • Triple Negative Breast Cancer
• ER/PR/Her and CK –ve
• 7 distinct molecular subtypes
• Basal-like 1(BL1), basal-like 2 (BL2), mesenchymal (M), mesenchymal stem-like (MSL),
immunomodulatory (IM), luminal androgen receptor(LAR), and unstable subgroups
• Different TNBC subgroups have different response to NACT
• Rate of pathological complete response(pCR) is better for BL1 comparatively

7. Prognostic gene signatures
• To identify patients who will benefit from adjuvant chemotherapy
• 1st Generation : Oncotype Dx and Mammaprint
• Better predictive power for early recurrences
• 2nd Generation : Prosigna/ Endopredict/ Breast Cancer Index
• Can predict both early and late recurrences
• Restricted to ER positive cancers, ER negative cases considered ‘high risk’
• Tests can be performed on formalin-fixed paraffin-embedded (FFPE)
samples

8. • Oncotype Dx
• RT- PCR assay, evaluates expression of
21 genes (16+5)
• Computes recurrence score from 0-
100 , low risk ( <18), intermediate risk
(18-30), high risk (>31)
• Validated by TAILORx study
• Benefit of addition of chemotherapy
to ER +ve, Her –ve, node –ve patients
treated with tamoxifen

9. • Mammaprint
• DNA microarray based test
• Evaluates expression of 70 genes
• Validated by MINDACT trial
• For patients < 61 yrs of age, with
stage 1/2 ER +ve, node –ve
breast cancer

10. • Prosigna
• RT-PCR based assay , using Nanostring technology
• Evaluates 50 genes
• Computes risk of recurrence (ROR) at 10 years
• Used in post menopausal women, Stage 1/2 , ER +ve on AI
• Endopredict
• RT-PCR
• Evaluates 8 cancer related genes
• Can be combined with tumor size and nodal status to give comprehensive risk
score Epclin
• Validated by ABCSG -6 trial

11. • Breast Cancer Index
• RT-PCR based
• Quantifies expression ratio of HOXB13 and IL17BR
• Integrated with Molecular Grade Index (MGI)
• Validated by Stockholm trial
• 8th edition of AJCC recommended to include Oncotype Dx in staging
patients with ER +ve, HER 2 –ve cancers

12. In situ hybridization (ISH)
• It’s a molecular technique that allows visualization of genes on a glass
slide
• Combines molecular genetics with traditional pathology
• Three methods of ISH
• Flourescent in situ hybridization (FISH)
• Chromogenic in situ hybridization (CISH)
• Silver in situ hybridization (SISH)

13. • In diagnostic breast pathology it enables
• HER 2 amplification
• Predicts response to anti HER 2 therapy and Chemotherapy in adjuvant, neoadjuvant
and metastatic settings
• FGFR1 amplification
• Predicts response to FGFR1 tyrosine kinase inhibitors
• Detection of fusion genes in rare histologic types
• MYB/MYBL1 amplification in Breast Adenoid cystic carcinoma

14. HER2 assesment
• HER2 , member of human epidermal growth factor
• Expressed in 15-20% of breast cancers
• Evolution of targeted therapy
• Trastuzumab/ pertuzumab : humanized monoclonal anti – HER2 antibody
• Lapatinib : HER2 tyrosine kinase inhibitor
• Trastuzumab Emtansine (T-DM1) : Drug delivery system

15. • ASCO/CAP guideline 2013
recommend
• Immunohistochemistry followed by
In situ hybridization in equivocal
cases
• Trastuzumab given to IHC 3+ and /or
ISH positive

16. • ‘HER2 double equivocal’ cancers are typically ER +ve and defined as
luminal B-like ( show high Ki67 indices)
• Oncologist may consider offering anti HER2 therapy based on patients
performance status and wish
• Caution :
• Residual carcinomas following taxane based chemotherapy may present giant
syncytial multinucleated looking cells harbouring abnormally high HER2
signals
• But HER2/CEP 17 ratio is normal

17. • Also , only a proportion of HER2 positive cases respond to anti HER2
therapy
• Most show progression within 1 year
• 15% patients receiving adjuvant therapy develop metastasis
• 50% patients receiving neoadjuvant therapy have residual diease following
treatment

18. Familial Breast Cancer
• Hereditary breast cancer < 10 % of all
• Half contain BRCA1 or BRCA2 mutations (tumor suppressor)
• BRCA1 : high grade features on histology
• Grade 3
• ductal NST
• medullary or atypical medullary
• pushing borders
• lymphocytic infiltrates
• Necrosis
• 80% are ER/PR/Her –ve , express basal markers ( CK5/6, p-cadherins, EGFR)
• BRCA testing should be offered to patients with above features

19. • BRCA2 have high grade features but are frequently ER +ve
• Inactivation of BRCA1 or BRCA2 leads to deficiency in homologous
recombination repair of DNA double-strand breaks and inter-strand
crosslinks
• More sensitive to cross linking agents (platinum salts) than spindle poisons
• Sensitive to PARP enzyme inhibitors ( block base excision repair pathway ) :
OLAPARIB

20. • BRCAness :
• tumours that have not arisen from a germline BRCA1 or BRCA2 mutation but
share certain molecular features, in particular a homologous recombination
defect
• TNBC
• Sensitive to PARP inhibitors

21. • PALB2 : partner and localizer of BRCA2
• Tumor suppressor
• 35% risk of breast cancer by age 70
• Increased susceptibility to PARP inhibitors

24. Intratumoral heterogeneity and liquid biosy
• Tumours are composed of multiple clones with distinct genetic alterations
• May result in the emergence of resistant cell populations upon therapeutic
pressure
• Metastatic outgrowths might stem from a minor cell subpopulation of the
primary tumour
• Traditional approaches in which DNA from the bulk of the tumour is
sequenced, do not have the power to detect minor subclones

25. • Liquid Biopsy : study of circulating tumor free DNA (cfDNA) and circulating
tumor cells (CTC)
• Can detect mutations present in metastatic foci and absent in primary
tumor
• ESR1 mutations : mediate resistance to oestrogen deprivation
• Found in higher frequency in metastatic breast cancer
• SoFEA trial : patients with ESR1 mutation showed better response to
fulvestrant (SERD) than exemestane (AI)

26. • cfDNA analysis can detect somatic reversion mutations in BRCA1 and
BRCA2 carriers with metastatic breast cancer treated with platinum
and/or PARP inhibitors
• Liquid biopsy can guide therapy in both early and metastatic breast
cancere.

27. References
• DeVita : principles of oncology (10th ed)
• Molecular diagnosis in breast cancer : F Pareja, C marchio

28. THANK YOU