High grade triple negative breast cancers. Gary Tse Department of Anatomical and Cellular Pathology Prince of Wales Hospital Hong Kong

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1 High grade triple negative breast cancers Gary Tse Department of Anatomical and Cellular Pathology Prince of Wales Hospital Hong Kong

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3 TNBC Heterogeneous group IDC NST high grade Metaplastic carcinoma Carcinoma with medullary features Apocrine carcinoma Salivary gland tumors secretory, adenoid cystic

4 Histological types of TNBC Geyer FC et al 2017 Am J Path 187:2139

5 Geyer FC et al 2017 Am J Path 187:2139

6 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:

7 Masuda H et al. Clin Cancer Res 2013;19: BLIS: Worst DFS and DSS BLIA: Good prognosis Burstein MD et al. Clin Cancer Res 2015;21:

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9 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

10 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)

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13 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

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15 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

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17 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

18 EMT markers 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 In vitro functional markers Increased migration Increased invasion Increased scattering Elongation of cell shape Resistance to anoikis

19 Model of the human mammary epithelial hierarchy linked to cancer subtypes

20 EMT in breast cancer Micro-metastasis and circulating tumor cell Claudin low breast cancer

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24 Carcinoma with medullary features Specific phenotypic characteristics Rounded tumor border Lymphocytic infiltrate High nuclear grade Brisk mitoses Geographic necrosis Usually triple negative

25 Correlation of TIL with molecular subtypes LI Lo Hi Total P-value Luminal A (HR+, HER2- and Ki67lo) (15.8%) 247 p<0.001 Luminal B (HR+, HER2+ and/ki67hi) (38.8%) 191 HER2-OE (HR-, HER2+) (70.3%) 37 (TNBC) (HR-, HER2-) (32) (23) (41.8%) (55) BLBC (HR-, HER2-, CK5/6+ and/egfr+) (54.2%) 24 Unclassified (HR-, HER2-, CK5/6-, EGFR-) (32.2%) 31 Total (30.6%) 530 High TIL more prevalent in aggressive subtypes of breast cancers Tsang J et al, 2014 Breast Cancer Res Treat 143:1

26 Cells in tumor microenvironment

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28 Paradoxical ROLES of Immune system in Cancer development

29 Roles of TIL Prognosis Outcome predictor for adjuvant therapy Outcome predictor for neoadjuvant therapy

30 Metaanalysis of prognostic value of TIL in TNBC Ibrahim E et al 2014 Breast Cancer Res Treat 148:467

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

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

33 Roles of TIL Prognosis Outcome predictor for adjuvant therapy Outcome predictor for neoadjuvant therapy

34 Prognostic role of stil in adjuvant trials Favorable effects of stil on TNBC and HER2+ cancers Savas P et al Nature Rev Clin Oncol 13:228

35 TILs association with survival -50% cutoff TNBC Greatest clinical benefit was found in cases with >50% TIL (Lymphocyte predominant breast cancer; LPBC) They are more prevalent in HER2+ and TNBC Pruneri G et al 2016 Ann Oncol 27:249

36 Roles of TIL Prognosis Outcome predictor for adjuvant therapy Outcome predictor for neoadjuvant therapy

37 TIL correlated with response to neoadjuvant chemotherapy 102 Stage II-III TNBC Neoadjuvant anthracyclinebased regimens alone, taxane based regimens alone or anthracycline and taxane sequentially or concurrently Av FU= 64.8 months Onon Met al 2012 Breast Cancer Res Treat 132:793

38 Methods for TIL evaluation H&E evaluation IHC staining Not feasible for a large cohort with limited tissues available; Sample collected may not be suitable Flow cytometry Multispectral imaging Gene expression profiling CD3 CD20 CD68 CD208

39 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

40 Different methods of H&E evaluation Quantitation Semi-quantitative TIL counting Number in hotspots Average number in whole section Location Intra-tumoral Stromal

41 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 ) 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 Ann Oncol 26:

42 Morphology, definitions, biological and diagnostic relevance of the different immune infiltrates found in breast cancer. Salgado R et al Ann Oncol 26:

43 Standardized approach for TILs evaluation in breast cancer Salgado R et al Ann Oncol 26:

44 Stromal TILs assessment Report average of the stromal area, do not focus on hotspot For intermediate group, evaluate different areas at higher magnification. Salgado R et al Ann Oncol 26:

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47 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

48 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

49 BRCA1 Located at 17q , 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

50 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

51 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

52 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 Oncol Kaeami F & Mehdipour P 2013 BioMed Res Int

53 Patient criteria for BRCA genes testing patients 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

54 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

55 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

56 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+

57 BRCA-directed therapy PARP inhibitor Platinum agent

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59 Summary : Molecular landscape of TNBC

60 TNBC subtypes and clinical relevance Denkert C et al 2017 Lancet

61 Thank you!