**2.2 TNBC subtypes**

In 2011, Lehmann et al. performed gene expression profiling of tumor samples from 587 TNBC patients and divided TNBC into six subtypes: [9, 11]

1.Basal-like-1 (BL-1):

	- a. Highly activated cell migration-related signaling pathways, extracellular matrix-receptor interaction pathways, and differentiation pathways (Wnt pathway, anaplastic lymphoma kinase pathway, transforming growth factor (TGF)-β signaling)
	- b. The M subtype has sarcoma-like or squamous epithelial cell-like tissue characteristics
	- c. Prone to develop resistance to chemotherapeutic drugs
	- a. Characterized by increased expression of immune cell-associated genes and pathways such as the Th1/Th2 pathway, NK cell pathway, B cell receptor signaling pathway, dendritic cell (DC) pathway, T cell receptor signaling, interleukin (IL)-12 pathway, and IL-7 pathway.
	- a. Although the LAR subtype does not express ER receptors, it does have highly activated hormonal-related signaling pathways (including steroid synthesis, porphyrin metabolism, and androgen/estrogen metabolism).
	- b. ESR1 (the gene encoding ERα) and other estrogen-regulated genes (PGR, FOXA, XBP1, GATA3) are present on micro-array profiling. Thus, there is molecular evidence of ER activation. However, they may be classified as "ER-negative" because <1% of these tumor cells express low levels of ER protein on IHC analysis
	- c. Androgen receptor (AR) is highly expressed (mRNA level is nine times) as well as high expression of AR on IHC (10 times)

This classification was validated by Masuda et al. [13]. They further compared the TNBC subtypes between the PAM50 basal-like subtype and non-basal-like subtypes (other subtypes grouped). All tumors in the BL1 and BL2 subtypes belonged to the basal-like PAM50 subtype, and most tumors in the LAR subtype belonged to the nonbasal-like PAM50 group. In the non-basal-like group, there were only three TNBC subtypes, LAR, MSL, and M; most of these tumors were the LAR subtype (59%). They further found that even though BL-1 and BL-2 are highly proliferative tumors, the BL-1 subtype had the highest pathological complete response (pCR) rate, and the BL2 subtype had the lowest pCR rate. Similarly, consistent with LAR's low pCR rate, the luminal A and B intrinsic subtypes, hormonally regulated tumors, showed less response to chemotherapy. Therefore, the LAR group had delayed recurrences compared with the other groups and did not have the lowest OS rate despite having a low pCR rate.

Burstein et al. distinguished TNBC subtypes into four types only: luminal-AR (LAR), mesenchymal (MES), basal-like immune-suppressed (BLIS), and basal-like immune-activated (BLIA) [14]. However, in an updated analysis, Lehman et al. reported that transcripts in the previously described IM and MSL subtypes were contributed from infiltrating lymphocytes and tumor-associated stromal cells, respectively [15]. Therefore, in their new refined classification, TNBC molecular subtypes were reduced from six (TNBCtype-6) to four (TNBCtype-4) tumor-specific subtypes (BL1, BL2, M, and LAR) (**Table 1**). PAM50 subtype "calls" distribution among the TNBC subtypes showed that most BL1, BL2, and M were basal-like, while LAR was enriched in HER2 and luminal subtypes.

#### **2.3 Benefits of sub-classifying TNBC**

	- 1.Age: Non-basal TNBC was reported in older patients than basal TNBC. LAR subtype was diagnosed in women of older age than all other subtypes.


#### **Table 1.**

*Different classification of subtypes of TNBC.*

	- 1.Basal Like: Cells have a complex DNA damage response and repair mechanisms to maintain genomic integrity. The most deleterious lesion, double-strand breaks are repaired by either HR (homologous recombination) or non-homologous end joining. Patients with mutations in the breast cancer susceptibility proteins BRCA1 and BRCA2A have cancers due to deficiency in HR repair. Moreover, these are dependent on other DNA repair mechanisms, the most prominent of which is the peroxisome proliferator-activated receptor (PARP)-based. Therefore, PARP inhibitors have significant antitumor effects on BRCA1/2-deficient tumors, and the inhibition effect on BRCA1-mutant tumors is 100–1000 times higher than


#### **Table 2.**

*Molecular pathways in TNBC subtypes as therapeutic targets.*

in tumors without such mutations [4]. The basal-like subgroup has increased expression of proliferation-related genes and DNA repair genes; therefore, they may be sensitive to anti-mitotic drugs such as taxanes and platinum and PARP inhibitors such as olaparib and veliparib.

