Inhibition of the HER2/ERBB2 receptor is a keystone to treating HER2-positive malignancies, particularly breast cancer, but a significant fraction of HER2-positive (HER2+) breast cancers recur or fail to respond. Anti-HER2 monoclonal antibodies, like trastuzumab or pertuzumab, and ATP active site inhibitors like lapatinib, commonly lack durability because of adaptive changes in the tumor leading to resistance. HER2+ cell line responses to inhibition with lapatinib were analyzed by RNAseq and ChIPseq to characterize transcriptional and epigenetic changes. Motif analysis of lapatinib-responsive genomic regions implicated the pioneer transcription factor FOXA1 as a mediator of adaptive responses. Lapatinib in combination with FOXA1 depletion led to dysregulation of enhancers, impaired adaptive upregulation of HER3, and decreased proliferation. HER2-directed therapy using clinically relevant drugs (trastuzumab with or without lapatinib or pertuzumab) in a 7-day clinical trial designed to examine early pharmacodynamic response to antibody-based anti-HER2 therapy showed reduced FOXA1 expression was coincident with decreased HER2 and HER3 levels, decreased proliferation gene signatures, and increased immune gene signatures. This highlights the importance of the immune response to anti-HER2 antibodies and suggests that inhibiting FOXA1-mediated adaptive responses in combination with HER2 targeting is a potential therapeutic strategy.

The identification of prognostic markers in patients receiving neoadjuvant therapy is crucial for treatment optimization in HER2-positive breast cancer, with the immune microenvironment being a key factor. Here, we investigate the complexity of B and T cell receptor (BCR and TCR) repertoires in the context of two phase III trials, NeoALTTO and CALGB 40601, evaluating neoadjuvant paclitaxel with trastuzumab and/or lapatinib in women with HER2-positive breast cancer. BCR features, particularly the number of reads and clones, evenness and Gini index, are heterogeneous according to hormone receptor status and PAM50 subtypes. Moreover, BCR measures describing clonal expansion, namely evenness and Gini index, are independent prognostic factors. We present a model developed in NeoALTTO and validated in CALGB 40601 that can predict event-free survival (EFS) by integrating hormone receptor and clinical nodal status, breast pathological complete response (pCR), stromal tumor-infiltrating lymphocyte levels (%) and BCR repertoire evenness. A prognostic score derived from the model and including those variables, HER2-EveNT, allows the identification of patients with 5-year EFS > 90%, and, in those not achieving pCR, of a subgroup of immune-enriched tumors with an excellent outcome despite residual disease.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer is a particularly aggressive form of the disease, and ultimately progresses in patients with metastases on standard therapies. Anthracyclines, such as doxorubicin, are an effective treatment for HER2-positive breast cancer, particularly when administered in combination with trastuzumab - however, doxorubicin-related cardiotoxicity has limited its use. Many patients are therefore never treated with anthracyclines, even upon disease progression, despite the potential for benefit. MM-302 is a novel, HER2-targeted antibody-liposomal doxorubicin conjugate that specifically targets HER2-overexpressing cells. Preclinical and Phase 1 data suggest that MM-302, as a monotherapy or in combination with trastuzumab, could be effective for managing previously treated, anthracycline-naïve, HER2-positive breast cancer, without the cardiotoxicity observed with free doxorubicin formulations.

MET expression and activation appear to be important for initiation and progression of triple-negative breast cancer. Tivantinib (ARQ 197) is an orally administered agent that targets MET, although recent preclinical data suggests the agent may have mechanisms of action that are independent of MET signaling. We conducted a phase 2 study of tivantinib monotherapy in patients with metastatic triple-negative breast cancer.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are fundamental drivers of the cell cycle and are required for the initiation and progression of various malignancies. Pharmacological inhibitors of CDK4/6 have shown significant activity against several solid tumours. Their primary mechanism of action is thought to be the inhibition of phosphorylation of the retinoblastoma tumour suppressor, inducing G1 cell cycle arrest in tumour cells. Here we use mouse models of breast carcinoma and other solid tumours to show that selective CDK4/6 inhibitors not only induce tumour cell cycle arrest, but also promote anti-tumour immunity. We confirm this phenomenon through transcriptomic analysis of serial biopsies from a clinical trial of CDK4/6 inhibitor treatment for breast cancer. The enhanced anti-tumour immune response has two underpinnings. First, CDK4/6 inhibitors activate tumour cell expression of endogenous retroviral elements, thus increasing intracellular levels of double-stranded RNA. This in turn stimulates production of type III interferons and hence enhances tumour antigen presentation. Second, CDK4/6 inhibitors markedly suppress the proliferation of regulatory T cells. Mechanistically, the effects of CDK4/6 inhibitors both on tumour cells and on regulatory T cells are associated with reduced activity of the E2F target, DNA methyltransferase 1. Ultimately, these events promote cytotoxic T-cell-mediated clearance of tumour cells, which is further enhanced by the addition of immune checkpoint blockade. Our findings indicate that CDK4/6 inhibitors increase tumour immunogenicity and provide a rationale for new combination regimens comprising CDK4/6 inhibitors and immunotherapies as anti-cancer treatment.

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate comprising trastuzumab and DM1, a microtubule polymerization inhibitor, covalently bound via a stable thioether linker. To characterize the pharmacokinetics (PK) of T-DM1 in patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer, data from four studies (TDM3569g, TDM4258g, TDM4374g, and TDM4688g) of single-agent T-DM1 administered at 3.6 mg/kg every 3 weeks (q3w) were assessed in aggregate.

Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated.

Treatment options for triple-negative breast cancer remain limited. Activation of the PI3K pathway via loss of PTEN and/or INPP4B is common. Buparlisib is an orally bioavailable, pan-class I PI3K inhibitor. We evaluated the safety and efficacy of buparlisib in patients with metastatic triple-negative breast cancer.

Human epidermal growth factor receptor 3 (HER3) is broadly expressed in breast cancer; high expression is associated with an adverse prognosis. Patritumab deruxtecan (HER3-DXd) is an investigational HER3-targeted antibody-drug conjugate that is being evaluated as a novel treatment in HER3-expressing advanced breast cancer in the U31402-A-J101 study.

Assaying for large numbers of low-frequency mutations requires sequencing at extremely high depth and accuracy. Increasing sequencing depth aids the detection of low-frequency mutations yet limits the number of loci that can be simultaneously probed. Here we report a method for the accurate tracking of thousands of distinct mutations that requires substantially fewer reads per locus than conventional hybrid-capture duplex sequencing. The method, which we named MAESTRO (for minor-allele-enriched sequencing through recognition oligonucleotides), combines massively parallel mutation enrichment with duplex sequencing to track up to 10,000 low-frequency mutations, with up to 100-fold fewer reads per locus. We show that MAESTRO can be used to test for chimaerism by tracking donor-exclusive single-nucleotide polymorphisms in sheared genomic DNA from human cell lines, to validate whole-exome sequencing and whole-genome sequencing for the detection of mutations in breast-tumour samples from 16 patients, and to monitor the patients for minimal residual disease via the analysis of cell-free DNA from liquid biopsies. MAESTRO improves the breadth, depth, accuracy and efficiency of mutation testing by sequencing.

We aimed to improve efficacy and reduce toxicity of high-risk human epidermal growth factor receptor 2 (HER2)-positive early breast cancer (EBC) treatment by replacing taxanes and trastuzumab with trastuzumab emtansine (T-DM1).

Despite the availability of multiple human epidermal growth factor receptor 2-targeted (HER2-targeted) treatments, therapeutic resistance in HER2+ breast cancer remains a clinical challenge. Intratumor heterogeneity for HER2 and resistance-conferring mutations in the PIK3CA gene (encoding PI3K catalytic subunit α) have been investigated in response and resistance to HER2-targeting agents, while the role of divergent cellular phenotypes and tumor epithelial-stromal cell interactions is less well understood. Here, we assessed the effect of intratumor cellular genetic heterogeneity for ERBB2 (encoding HER2) copy number and PIK3CA mutation on different types of neoadjuvant HER2-targeting therapies and clinical outcome in HER2+ breast cancer. We found that the frequency of cells lacking HER2 was a better predictor of response to HER2-targeted treatment than intratumor heterogeneity. We also compared the efficacy of different therapies in the same tumor using patient-derived xenograft models of heterogeneous HER2+ breast cancer and single-cell approaches. Stromal determinants were better predictors of response than tumor epithelial cells, and we identified alveolar epithelial and fibroblastic reticular cells as well as lymphatic vessel endothelial hyaluronan receptor 1-positive (Lyve1+) macrophages as putative drivers of therapeutic resistance. Our results demonstrate that both preexisting and acquired resistance to HER2-targeting agents involve multiple mechanisms including the tumor microenvironment. Furthermore, our data suggest that intratumor heterogeneity for HER2 should be incorporated into treatment design.

Using transgenic mouse models, cell line-based functional studies, and clinical specimens, we show that cyclin D1/CDK4 mediate resistance to targeted therapy for HER2-positive breast cancer. This is overcome using CDK4/6 inhibitors. Inhibition of CDK4/6 not only suppresses Rb phosphorylation, but also reduces TSC2 phosphorylation and thus partially attenuates mTORC1 activity. This relieves feedback inhibition of upstream EGFR family kinases, resensitizing tumors to EGFR/HER2 blockade. Consequently, dual inhibition of EGFR/HER2 and CDK4/6 invokes a more potent suppression of TSC2 phosphorylation and hence mTORC1/S6K/S6RP activity. The suppression of both Rb and S6RP enhances G1 arrest and a phenotype resembling cellular senescence. In vivo, CDK4/6 inhibitors sensitize patient-derived xenograft tumors to HER2-targeted therapies and delay tumor recurrence in a transgenic model of HER2-positive breast cancer.

Brain metastases represent the greatest clinical challenge in treating HER2-positive breast cancer. We report the development of orthotopic patient-derived xenografts (PDXs) of HER2-expressing breast cancer brain metastases (BCBM), and their use for the identification of targeted combination therapies. Combined inhibition of PI3K and mTOR resulted in durable tumor regressions in three of five PDXs, and therapeutic response was correlated with a reduction in the phosphorylation of 4EBP1, an mTORC1 effector. The two nonresponding PDXs showed hypermutated genomes with enrichment of mutations in DNA-repair genes, which suggests an association of genomic instability with therapeutic resistance. These findings suggest that a biomarker-driven clinical trial of PI3K inhibitor in combination with an mTOR inhibitor should be conducted for patients with HER2-positive BCBM.

Oncoproteins and tumor suppressors antagonistically converge on critical nodes governing neoplastic growth, invasion, and metastasis. We discovered that phosphorylation of the ETS1 and ETS2 transcriptional oncoproteins at specific serine or threonine residues creates binding sites for the COP1 tumor suppressor protein, which is an ubiquitin ligase component, leading to their destruction. In the case of ETS1, however, phosphorylation of a neighboring tyrosine residue by Src family kinases disrupts COP1 binding, thereby stabilizing ETS1. Src-dependent accumulation of ETS1 in breast cancer cells promotes anchorage-independent growth in vitro and tumor growth in vivo. These findings expand the list of potential COP1 substrates to include proteins whose COP1-binding sites are subject to regulatory phosphorylation and provide insights into transformation by Src family kinases.

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

Evidence-based treatments for metastatic, human epidermal growth factor receptor 2 (HER2)-positive breast cancer to the CNS are limited. We previously reported modest activity of neratinib monotherapy for HER2-positive breast cancer brain metastases. Here we report the results from additional study cohorts.

In contrast to recurrence after initial diagnosis of stage I-III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS).

DESTINY-Breast01 (NCT03248492) evaluated trastuzumab deruxtecan (T-DXd; DS-8201) in patients with heavily pretreated HER2-positive metastatic breast cancer (mBC). We present a subgroup of 24 patients with a history of treated brain metastases (BM), a population with limited treatment options. In patients with BMs, the confirmed objective response rate (cORR) was 58.3% [95% confidence interval (CI), 36.6%-77.9%], and the median progression-free survival (mPFS) was 18.1 months (95% CI, 6.7-18.1 months). In patients without BMs (n = 160), cORR was 61.3% and mPFS was 16.4 months. Eight patients (47.1%) experienced a best overall intracranial response of partial response or complete response. Seven patients (41.2%) had a best percentage change in brain lesion diameter from baseline consistent with stable disease. Two patients (8.3%) with BMs and two (1.3%) without BMs experienced progression in the brain. The safety profile of T-DXd was consistent with previous studies. The durable clinical activity of T-DXd in this population warrants further investigation.

Triple-negative breast cancer (TNBC) is a heterogeneous and clinically aggressive disease for which there is no targeted therapy. BET bromodomain inhibitors, which have shown efficacy in several models of cancer, have not been evaluated in TNBC. These inhibitors displace BET bromodomain proteins such as BRD4 from chromatin by competing with their acetyl-lysine recognition modules, leading to inhibition of oncogenic transcriptional programs. Here we report the preferential sensitivity of TNBCs to BET bromodomain inhibition in vitro and in vivo, establishing a rationale for clinical investigation and further motivation to understand mechanisms of resistance. In paired cell lines selected for acquired resistance to BET inhibition from previously sensitive TNBCs, we failed to identify gatekeeper mutations, new driver events or drug pump activation. BET-resistant TNBC cells remain dependent on wild-type BRD4, which supports transcription and cell proliferation in a bromodomain-independent manner. Proteomic studies of resistant TNBC identify strong association with MED1 and hyper-phosphorylation of BRD4 attributable to decreased activity of PP2A, identified here as a principal BRD4 serine phosphatase. Together, these studies provide a rationale for BET inhibition in TNBC and present mechanism-based combination strategies to anticipate clinical drug resistance.