Table 1. Consensus Signature components based on putative steps required for effective anthracycline-induced cytotoxicity.

Step	Surrogate marker	Association with pCR	Rationale
Penetration of drug into the tumor bed	SHARP1 signature Hypoxia signature (HIF)	Negative Negative	Hypoxia, promoted by HIFs, is a well-known contributor to decreased drug penetration, and chemoresistance.1 Montagner et al. recently described a hypoxia signature of 22 genes, with increased expression correlated with increased HIF activity.2 A direct interaction between SHARP1 (a downstream target of the tumor suppression gene p63) and HIF1α and HIF2α was demonstrated, with a signature of low SHARP1 activity in TNBC conferring increased HIF function and increased hypoxia.2 With the SHARP1 signature measuring low SHARP activity and thus increased HIF function, it has a negative association with pCR.
Location of topoIIα protein within the nucleus	LAPTM4B	Negative	In order to work effectively, the target of anthracyclines, topoIIα protein, must have access to nuclear DNA; thus, it must be located in the nucleus. Nuclear export of topoIIα protein may contribute to anthracycline resistance.3,4 topoIIα protein nuclear location might be inferred using the expression level of LAPTM4B.5 LAPTM4B gene resides on chromosome 8q22, with overexpression shown to increase sequestration of anthracyclines in the cytoplasm. Increased levels of LAPTM4B mRNA have been correlated with increased anthracycline resistance, whereas selective depletion of LAPTM4B significantly increased sensitivity to anthracycline, but not cisplatin or taxane, chemotherapy.5
Increased expression of topoIIα mRNA, independent of proliferation	topoIIα mRNA topoIIα mRNA: AURKA topoIIα mRNA: AURKA signature	Positive Positive Positive	TOP2A transcription can be enhanced by proliferation signals independently of gene aberrations and topoIIα protein is strongly influenced by proliferation.6 Increased expression of topoIIα protein therefore may be seen in the setting of highly proliferating tumors, without correlating with an increased likelihood of response specifically to anthracyclines. By determining the ratio of expression of topoIIα mRNA relative to that of a known proliferation marker (Aurora kinase A gene, AURKA,7,8 or AURKA gene signature), tumors with increased topoIIα mRNA independent of proliferation might be determined.
Induction of apoptosis	YWHAZ Minimal gene signature (MS)	Negative Positive	The anti-apoptotic gene YWHAZ (coding for 14-3-3ζ) resides on chromosome 8q22 close to LAPTM4B gene and may promote de novo anthracycline resistance.5 Increased expression has been associated with increased doxorubicin resistance in breast cancer cell lines, and early relapses after anthracycline chemotherapy. siRNA knockdown of YWHAZ in breast cancer cell lines significantly increased doxorubicin-induced apoptosis.5 An alternate marker of apoptosis is the MS,9 comprising two genes, SHARP1 and CCNG2. As with SHARP1, CCNG2 is a downstream target of p63. As p63 is inhibited by mutant p53, lack of MS expression implies dysfunction in the p53 pathway, the major apoptotic pathway in the presence of oncogenic stress, and may be a suitable surrogate for lack of apoptosis.
Active immune function	Immune function signature (STAT1) Stromal signature (PLAU)	Positive Negative	Both innate and adaptive immune responses are important in anthracycline toxicity.10–13 Anthracyclines trigger immunogenic cell death by eliciting tumor-specific IFNγ CD8+ cytotoxic T lymphocytes, thus an anthracycline-induced anticancer immune response can help eradicate residual cancer cells, or maintain residual cells in state of dormancy. Moreover, immune module scores14,15 have been associated with higher probability of achieving pCR after anthracycline±taxane chemotherapy among all breast cancer subtypes when defined by immunohistochemistry.16 Closely related to immune function, stromal signatures may also be useful in predicting anthracycline sensitivity or resistance.15,17

Abbreviations: HIF, hypoxia-inducible factor; IFNγ, interferon gamma; LAPTM4B, lysosomal-associated protein transmembrane 4B gene; mRNA, messenger RNA; MS, minimal gene signature; pCR, pathological complete response; siRNA, small interfering RNA; TNBC, triple-negative breast cancer.

References for this table are listed in Supplementary Materials.