high grade triple negative breast cancers...• ashkenazi jewish heritage and breast cancer at any...

High grade triple negative breast cancers

Gary TseDepartment of Anatomical and Cellular PathologyPrince of Wales HospitalHong Kong

TNBC

• Heterogeneous group

• IDC NST high grade• Metaplastic carcinoma• Carcinoma with medullary features• ‘Apocrine’ carcinoma

• ‘Salivary gland’ tumors – secretory, adenoid cystic

Histological types of TNBC

Geyer FC et al 2017 Am J Path 187:2139

Geyer FC et al 2017 Am J Path 187:2139

TNBC – sub-classification

mRNA profiling mRNA and DNA profiling

- Luminal AR (LAR)- Mesenchymal (MES)- Basal-like immunosuppressed (BLIS)- Basal-like immune activated (BLIA)

Lehmann BD, et al. J Clin Invest.2011; 121: 2750-67

BLIS: Worst DFS and DSSBLIA: Good prognosis

Burstein MD et al. Clin Cancer Res 2015;21:1688-1698

Masuda H et al. Clin Cancer Res 2013;19:5533-5540

Metaplastic carcinoma

Classification

Low grade• Low grade adeno-squamous carcinoma• Fibromatosis like metaplastic carcinoma

High grade• Squamous cell carcinoma• Spindle cell carcinoma• Carcinoma with mesenchymal differentiation• Myoepithelial carcinoma

Squamous cell carcinoma

• Usually present as cystic lesion, lined by tumor cells of varying degree of pleomorphism

• Infiltration with stromal reaction• Inflammatory response is usually intense• Invasive front usually shows spindle cells (less squamous differentiation)• Acantholytic variant reminiscent of angiosarcoma• May be pure or mixed with NST• Need to rule out other primary SCC (esp skin)

Spindle cell carcinoma

• Atypical spindle cells in variable patterns, long or short fascicles• Usually moderate to severe nuclear pleomorphism• Focal areas of more cellular cohesion may be seen• Likely overlap with squamous with spindle carcinoma and

myoepithelial carcinoma• May show DCIS in the vicinity

MC with mesenchymal differentiation

• Often admixed with mesenchymal components (chondroid, osseous, rhabdomyoid or neuroglial)

• May be well differentiated or poorly differentiated (sarcoma like)• Carcinomatous component may appear as glandular tubules, solid

clusters or squamous differentiation

Epithelial-to-Mesenchymal transition (EMT)

• Cancer cells undergo phenotypic and molecular alterations representing mesenchymal differentiation or stem/ progenitor cell phenotypes

• Phenotypic changes• cuboidal/cobblestone morphology to

elongated, spindle, fibroblastic type morphology

• Molecular changes• loss of epithelial cell adhesion molecules

and acquisition of mesenchymal markers• EMT associated with stemness, basal lineage

and treatment response in breast cancer• Crucial event in the metastatic process

Granit RZ et al 2014 Wiley Interdiscip rev syst biol med

EMT markers• In vitro functional markers

• Increased migration• Increased invasion• Increased scattering• Elongation of cell shape• Resistance to anoikis

• Increased expression• N-cadherin• Vimentin• Snail (EMT TF)• Slug (EMT TF)• Twist (EMT TF)• FOXC2 (EMT TF)• MMP

• Decreased expression• E-cadherin• cytokeratin

• Increased nuclear accumulation b-catenin (Wnt signaling)• Smad-2/3 (TGF-b signaling)• NF-kB

• Snail• Slug• Twist

Model of the human mammary epithelial hierarchy linked to cancer subtypes

EMT in breast cancer

• Micro-metastasis and circulating tumor cell

• Claudin low breast cancer

Carcinoma with medullary features

• Specific phenotypic characteristics• Rounded tumor border

• Lymphocytic infiltrate

• High nuclear grade

• Brisk mitoses

• Geographic necrosis

• Usually triple negative

Correlation of TIL with molecular subtypes

LI

Lo Hi Total P-value

Luminal A (HR+, HER2- and Ki67lo) 208 39 (15.8%) 247 p<0.001

Luminal B (HR+, HER2+ and/Ki67hi) 117 74 (38.8%) 191

HER2-OE (HR-, HER2+) 11 26 (70.3%) 37

(TNBC) (HR-, HER2-) (32) (23) (41.8%) (55)

BLBC (HR-, HER2-, CK5/6+ and/EGFR+) 11 13 (54.2%) 24

Unclassified (HR-, HER2-, CK5/6-, EGFR-) 21 10 (32.2%) 31

Total 368 162 (30.6%) 530

High TIL more prevalent in aggressive subtypes of breast cancers

Tsang J et al, 2014 Breast Cancer Res Treat 143:1

Cells in tumor microenvironment

Paradoxical ROLES of Immune system in Cancer development

Roles of TIL

• Prognosis

• Outcome predictor for adjuvant therapy

• Outcome predictor for neoadjuvant therapy

Meta-analysis of prognostic value of TIL in TNBC

Ibrahim E et al 2014 Breast Cancer Res Treat 148:467

Meta-analysis of prognostic value of TIL in TNBC

• Included eight eligible studies with 2987 patients• Overall median FU of 113 months• Reduction in risk with high TIL

• Recurrence: 30% (HR=0.07)• Distant recurrence: 22% (HR=0.78)• Death: 34% (HR=0.66)

• 15-20% reduction of any events for every 10% TIL enrichment


• High TIL predict better survival regardless of its location and phenotyping

• Both HE and IHC method of TIL interpretation demonstrated prognostic value (Lower HR with IHC)


Roles of TIL

• Prognosis



Prognostic role of sTILin adjuvant trials

Savas P et al. 2016 Nature Rev Clin Oncol 13:228

Favorable effects of sTIL on TNBC and HER2+ cancers

TILs association with survival -50% cutoff

Pruneri G et al 2016 Ann Oncol 27:249

• Greatest clinical benefit was found in cases with >50% TIL (Lymphocyte predominant breast cancer; LPBC)

• They are more prevalent in HER2+ and TNBC

TNBC

Roles of TIL

• Prognosis



TIL correlated with response to neoadjuvant chemotherapy

• 102 Stage II-III TNBC

• Neoadjuvant anthracycline-based regimens alone, taxanebased regimens alone or anthracycline and taxanesequentially or concurrently

• Av FU= 64.8 months

Onon Met al 2012 Breast Cancer Res Treat 132:793

Methods for TIL evaluationH&E evaluation IHC staining

Flow cytometry Multispectral imaging Gene expression profiling

CD3CD20CD68CD208

Not feasible for a large cohort with limited tissues available; Sample collected may not be suitable

Methods for TILs evaluation: H&E staining

• Disadvantages• Non specific

• Does not delineate different lymphocyte subsets

• Advantages• Low cost

• Aligned with routine clinical pathology

• Comparative predictive value to immune gene expression profile

Denkert C et al 2015 J Clin Oncol

Different methods of H&E evaluation

• Quantitation• Semi-quantitative• TIL counting

• Number in hotspots• Average number in whole section

• Location• Intra-tumoral• Stromal

Proposed guidelines for TIL evaluation in breast cancer by International TILs working group

• TILs should be reported for the stromal compartment (=% stromal TILs) • i.e. area occupied by mononuclear inflammatory cells over total intratumoral stromal area, not the

number of stromal cells • TILs should be evaluated within the borders of the invasive tumor

• Exclude TILs outside of the tumor border and around DCIS and normal lobules• Exclude TILs in tumor zones with crush artifacts, necrosis, regressive hyalinization as well as in the

previous core biopsy site • All mononuclear cells (including lymphocytes and plasma cells) should be scored, but PMN are excluded• One section (4–5 µm, magnification ×200–400) per patient is currently considered to be sufficient• Full sections are preferred over biopsies whenever possible

• Cores can be used in the pre-therapeutic neoadjuvant setting• A full assessment of average TILs (not hotspot) in the tumor area by the pathologist should be used • TILs should be assessed as a continuous parameter• No formal recommendation for a clinically relevant TIL threshold(s) can be given at this stage

Salgado R et al. 2015 Ann Oncol 26:259-271

Morphology, definitions, biological and diagnostic relevance of the different

immune infiltrates found in breast cancer.


Standardized approach for TILs evaluation in breast

cancer


Stromal TILs assessment

• Report average of the stromal area, do not focus on hotspot

• For intermediate group, evaluate different areas at higher magnification.


Carcinoma with medullary features

• Specific phenotypic characteristics• Rounded tumor border

• Lymphocytic infiltrate

• High nuclear grade

• Brisk mitoses

• Geographic necrosis

• Usually triple negative

• BRCA1 associated tumor

DNA damage repair mechanisms

• Nucleotide excision repair (NER)• Distortion of the DNA double helix repaired• xeroderma pigmentosum-related genes (XPA, XPC etc.) and Cockayne's syndrome-related genes

(ERCC6 etc)

• Base excision report (BER)• Nonhelix-distorting nucleotide base lesions or single-strand breaks• Genes: AP endonuclease, DNA glycosylase

• Mismatch repair (MMR)• Incorrectly paired nucleotides, insertion and deletion loops repaired• Genes: MSH2, MLH1

• Non-homologous end joining (NHEJ)• Chromosomal break• Genes: ku70, ku80, DNA-PKcs, XRCC4

• Homologous recombination repair (HRR)• Single strand breaks and double strand breaks repaired• Gene: BRCA1/2, BRT1, BLM, RAD

• High degree of fidelity• Other mechanisms are more error

prone leading to genomic instability

BRCA1

• Located at 17q12-21.1, contained 24 exons and encoded a protein of 220 kD

• RING domain in the N-terminus• nteracts with BRCA1-associated RING domain protein 1

(BARD1), increases BRCA1 ubiquitin ligase activity

• BRCT domain in C-terminus• conserved sequence for DNA repair and cell cycle

• site for numerous phosphoprotein interactions.

• Region of BRCA1 between exons 11 and 13• Disordered structure

• 2 nuclear localization motifs

• binding site for a number of proteins (RB, c-Myc, DNA repair proteins RAD50 RAD51 and PALB2) involved in multiple cellular pathways.

Clark S et al 2012 Comput Struct Biotechnol J

BRCA1 network: beyond DNA repair

• DNA repair• Cell cycle progression

• Part of BASC (BRCA1 associated genome surveillance complex)

• Protein ubiquitination• Heterodimer with BARD1 as E3

ligase• Chromatin remodeling• Transcription regulation

• Co-activator: STAT1, p53 (derepressor)

• Co-repressor: c-Myc, ER

BRCA2

• Located at 13q12-13, composed of 27 exons and encoded a protein of 385 kD

• Identified by linkage analysis with BRCA1 but shows little homology with BRCA1 and other known genes

• A large part of protein with undefined domain

• Contains a N-terminus transactivation domain, a long exon 11 containing RAD51-specific binding site and a DNA binding domain toward the C-terminus

• Function mainly on HRR

Germline mutation in BRCA1/2• Mutations in BRCA1/2 accounts for 15% familial breast cancers• Higher frequency in Ashkenazi Jewish population due to the presence of founder mutations

• BRCA1.185delAG, BRCA1.5382insC, and BRCA2.6174delT accounts for 10% of familial cases

• Founder mutation and ethnic-group-specific mutations have been demonstrated in other populations • The most common types of mutation are attributed to small insertion/deletion frameshift, nonsynonymous

truncation, and disruption of splice site• Larger genomic rearrangements comprise 1/3 of all mutation in BRCA1• Higher rate of duplication/deletion in BRCA1 Vs BRCA2 (42% Vs 20%) due to accumulation of Alu sequences• The risk of cancer development depends on the position and type of mutation.

Shiovitz S & Korde L 2015 Ann OncolKaeami F & Mehdipour P 2013 BioMed Res Int

Patient criteria for BRCA genes testingpatients with personal history of breast cancer and one or more of the followings

• Age of onset less than or equal to 50• Triple negative tumor and age <= 60• Ashkenazi Jewish heritage and breast cancer at

any age• Two or more primary breast cancer (either

asynchronous, synchronous, bilateral, or multicentric)

• A first-degree relative with breast cancer diagnosed at age less than or equal to 50

• Two relatives on the same side of the family with breast cancer and/or pancreatic cancer

• Family or personal history of ovarian cancer, fallopian cancer, or primary peritoneal cancer

• Male breast cancer• Known mutation carrier in the family

patients without a personal history of breast cancer but with one or more of the followings

• First-degree or second-degree relative with age onset of breast cancer <= 45

• Ashkenazi Jewish heritage and family history of breast cancer at any age

• >= 2 primary breast cancer (either asynchronous, synchronous, bilateral, or multicentric) in a single family member

• >= 2 relatives on the same side of the family with breast and/or pancreatic cancer

• Family or personal history of ovarian, fallopian, or primary peritoneal cancer

• Male breast cancer• Known mutation carrier in the family

NCCN/ American Society of Breast Surgeon Guideline

BRCA-1 associated cancers and BLBC

• Features for BRCA1 associated tumor overlap with BLBC• BRCA2 associated cancer do not have a consistent distinct molecular phenotype as BRCA1 associated cancer,

but tend to be ER+ and HER2-• BRCA1 and BRCA2 associated tumor development follows different paths

• BRCA1 involved in more diverse cellular processes

Turner N et al 2004 Nature review cancer

BRCAness

• “BRCAness” was introduced in 2004

• ‘hallmarks’ of breast and ovarian cancer susceptibility to the known inherited BRCA1 and BRCA2 mutations in otherwise sporadic cancers, i.e. phenocopy of BRCA1 or BRCA2 mutation

• Phenotypes of HRR deficiency in the absence of a germline BRCA1/2 mutation • Exhibit extreme level of genomic instability: high frequency of deletion and reordered

chromosomes• Sensitive to drugs that cause DSB by inhibiting normal progression of replication fork, e.g.

platinum salts, PARP inihibitor

• Mechanisms:• BRCA1/2 downregulation by mechanisms other than mutation• Defective in other genes involved in HRR

• The concept is not limited to breast and ovarian cancer, but also other cancers, e.g. prostate cancer and pancreatic ductal adenocarcinoma

Mechanisms in BRCA1/2 inhibition

• BRCA1• Promoter methylation

• 15% of sporadic breast cancer; higher incidence in TNBC

• Deletion• Loss of 17q21• Homozygous deletion

• Increased ID4 (negative regulator of BRCA1) expression

• BRCA2• Rare promoter hypermethylation• Gene amplification of EMSY (also known as C11orf30) at chromosome 11

• EMSY inhibit BRCA2 transcriptional activity by interacting with exon 3 of BRCA2• EMSY amplification in upto 13% of sporadic breast cancer, mostly ER+

BRCA-directed therapy

• PARP inhibitor

• Platinum agent

Summary : Molecular landscape of TNBC

TNBC subtypes and clinical relevance

Denkert C et al 2017 Lancet

Thank you !

high grade triple negative breast cancers...• ashkenazi jewish heritage and breast cancer at any...

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