Jeremy Hoog

Genome remodelling in a basal-like breast cancer metastasis and xenograft.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumour progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumour, a brain metastasis and a xenograft derived from the primary tumour. The metastasis contained two de novo mutations and a large deletion not present in the primary tumour, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumour mutations and displayed a mutation enrichment pattern that resembled the metastasis. Two overlapping large deletions, encompassing CTNNA1, were present in all three tumour samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared with the primary tumour indicate that secondary tumours may arise from a minority of cells within the primary tumour.

Whole Genome Analysis Informs Breast Cancer Response to Aromatase Inhibition

To correlate the variable clinical features of oestrogen-receptor-positive breast cancer with somatic alterations, we studied pretreatment tumour biopsies accrued from patients in two studies of neoadjuvant aromatase inhibitor therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to haematopoietic disorders. Mutant MAP3K1 was associated with luminal A status, low-grade histology and low proliferation rates, whereas mutant TP53 was associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon aromatase inhibitor treatment. Pathway analysis demonstrated that mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in oestrogen-receptor-positive breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumour biology, but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

Randomized Phase II Neoadjuvant Comparison Between Letrozole, Anastrozole, and Exemestane for Postmenopausal Women With Estrogen Receptor–Rich Stage 2 to 3 Breast Cancer: Clinical and Biomarker Outcomes and Predictive Value of the Baseline PAM50-Based Intrinsic Subtype—ACOSOG Z1031

PURPOSE Preoperative aromatase inhibitor (AI) treatment promotes breast-conserving surgery (BCS) for estrogen receptor (ER)-positive breast cancer. To study this treatment option, responses to three AIs were compared in a randomized phase II neoadjuvant trial designed to select agents for phase III investigations. PATIENTS AND METHODS Three hundred seventy-seven postmenopausal women with clinical stage II to III ER-positive (Allred score 6-8) breast cancer were randomly assigned to receive neoadjuvant exemestane, letrozole, or anastrozole. The primary end point was clinical response. Secondary end points included BCS, Ki67 proliferation marker changes, the Preoperative Endocrine Prognostic Index (PEPI), and PAM50-based intrinsic subtype analysis. RESULTS On the basis of clinical response rates, letrozole and anastrozole were selected for further investigation; however, no other differences in surgical outcome, PEPI score, or Ki67 suppression were detected. The BCS rate for mastectomy-only patients at presentation was 51%. PAM50 analysis identified AI-unresponsive nonluminal subtypes (human epidermal growth factor receptor 2 enriched or basal-like) in 3.3% of patients. Clinical response and surgical outcomes were similar in luminal A (LumA) versus luminal B tumors; however, a PEPI of 0 (best prognostic group) was highest in the LumA subset (27.1% v 10.7%; P = .004). CONCLUSION Neoadjuvant AI treatment markedly improved surgical outcomes. Ki67 and PEPI data demonstrated that the three agents tested are biologically equivalent and therefore likely to have similar adjuvant activities. LumA tumors were more likely to have favorable biomarker characteristics after treatment; however, occasional paradoxical increases in Ki67 (12% of tumors with > 5% increase after therapy) suggest treatment-resistant cells, present in some LumA tumors, can be detected by post-treatment profiling.

Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts

To characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality when Combined with Estrogen Deprivation in Estrogen Receptor-Positive Breast Cancer

Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)-positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110alpha and p110beta, in ER(+) breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110alpha RNAi inhibited growth and promoted apoptosis in all tested ER(+) breast cancer cells under estrogen deprived-conditions, whereas p110beta RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110alpha/p110beta inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER(+) breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110alpha and p110beta catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER(+) breast cancer.

Targeting Chk1 in p53-deficient triple-negative breast cancer is therapeutically beneficial in human-in-mouse tumor models

Patients with triple-negative breast cancer (TNBC) - defined by lack of estrogen receptor and progesterone receptor expression as well as lack of human epidermal growth factor receptor 2 (HER2) amplification - have a poor prognosis. There is a need for targeted therapies to treat this condition. TNBCs frequently harbor mutations in TP53, resulting in loss of the G1 checkpoint and reliance on checkpoint kinase 1 (Chk1) to arrest cells in response to DNA damage. Previous studies have shown that inhibition of Chk1 in a p53-deficient background results in apoptosis [corrected] in response to DNA damage. We therefore tested whether inhibition of Chk1 could potentiate the cytotoxicity of the DNA damaging agent irinotecan in TNBC using xenotransplant tumor models. Tumor specimens from patients with TNBC were engrafted into humanized mammary fat pads of immunodeficient mice to create 3 independent human-in-mouse TNBC lines: 1 WT (WU-BC3) and 2 mutant for TP53 (WU-BC4 and WU-BC5). These lines were tested for their response to irinotecan and a Chk1 inhibitor (either UCN-01 or AZD7762), either as single agents or in combination. The combination therapy induced checkpoint bypass and apoptosis in WU-BC4 and WU-BC5, but not WU-BC3, tumors. Moreover, combination therapy inhibited tumor growth and prolonged survival of mice bearing the WU-BC4 line, but not the WU-BC3 line. In addition, knockdown of p53 sensitized WU-BC3 tumors to the combination therapy. These results demonstrate that p53 is a major determinant of how TNBCs respond to therapies that combine DNA damage with Chk1 inhibition.

Relationship between plasma estradiol levels and estrogen-responsive gene expression in estrogen receptor-positive breast cancer in postmenopausal women.

PURPOSE To determine whether plasma estradiol (E2) levels are related to gene expression in estrogen receptor (ER)-positive breast cancers in postmenopausal women. Materials and METHODS Genome-wide RNA profiles were obtained from pretreatment core-cut tumor biopsies from 104 postmenopausal patients with primary ER-positive breast cancer treated with neoadjuvant anastrozole. Pretreatment plasma E2 levels were determined by highly sensitive radioimmunoassay. Genes were identified for which expression was correlated with pretreatment plasma E2 levels. Validation was performed in an independent set of 73 ER-positive breast cancers. Results The expression of many known estrogen-responsive genes and gene sets was highly significantly associated with plasma E2 levels (eg, TFF1/pS2, GREB1, PDZK1 and PGR; P < .005). Plasma E2 explained 27% of the average expression of these four average estrogen-responsive genes (ie, AvERG; r = 0.51; P < .0001), and a standardized mean of plasma E2 levels and ER transcript levels explained 37% (r, 0.61). These observations were validated in an independent set of 73 ER-positive tumors. Exploratory analysis suggested that addition of the nuclear coregulators in a multivariable analysis with ER and E2 levels might additionally improve the relationship with the AvERG. Plasma E2 and the standardized mean of E2 and ER were both significantly correlated with 2-week Ki67, a surrogate marker of clinical outcome (r = -0.179; P = .05; and r = -0.389; P = .0005, respectively). CONCLUSION Plasma E2 levels are significantly associated with gene expression of ER-positive breast cancers and should be considered in future genomic studies of ER-positive breast cancer. The AvERG is a new experimental tool for the study of putative estrogenic stimuli of breast cancer.

CDK4/6 inhibition triggers anti-tumour immunity.

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.

ESR1 gene amplification in breast cancer: a common phenomenon?

Lindsay A Brown1,2, Jeremy Hoog3, Suet-Feung Chin4,5, Yu Tao3, Abd Alnaser Zayed1,2, Koei Chin6, Andrew E Teschendorff4,5, John F Quackenbush7, John C Marioni8, Samuel Leung9,10, Charles M Perou11, Torsten O Neilsen9,10, Matthew Ellis3, Joe W Gray6, Philip S Bernard7, David G Huntsman1,2,9,10,12, and Carlos Caldas4,5,12 1Center for Translational and Applied Genomics, and the British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4E6, Canada.

A Phase I Trial of BKM120 (Buparlisib) in Combination with Fulvestrant in Postmenopausal Women with Estrogen Receptor-Positive Metastatic Breast Cancer.

Purpose: This trial was conducted to determine the maximum tolerated dose (MTD) and preliminary efficacy of buparlisib, an oral pan-class I PI3K inhibitor, plus fulvestrant in postmenopausal women with metastatic estrogen receptor positive (ER+) breast cancer. Experimental Design: Phase IA employed a 3+3 design to determine the MTD of buparlisib daily plus fulvestrant. Subsequent cohorts (phase IB and cohort C) evaluated intermittent (5/7-day) and continuous dosing of buparlisib (100 mg daily). No more than 3 prior systemic treatments in the metastatic setting were allowed in these subsequent cohorts. Results: Thirty-one patients were enrolled. MTD was defined as buparlisib 100 mg daily plus fulvestrant. Common adverse events (AE) included fatigue (38.7%), transaminases elevation (35.5%), rash (29%), and diarrhea (19.4%). C-peptide was significantly increased during treatment, consistent with on-target effect of buparlisib. Compared with intermittent dosing, daily buparlisib was associated with more frequent early onset AEs and higher buparlisib plasma concentrations. Among the 29 evaluable patients, the clinical benefit rate was 58.6% (95% CI, 40.7%–74.5%). Response was not associated with PIK3CA mutation or treatment cohort; however, loss of PTEN, progesterone receptor (PgR) expression, or mutation in TP53 was most common in resistant cases, and mutations in AKT1 and ESR1 did not exclude treatment response. Conclusions: Buparlisib plus fulvestrant is clinically active with manageable AEs in patients with metastatic ER+ breast cancer. Weekend breaks in buparlisib dosing reduced toxicity. Patients with PgR negative and TP53 mutation did poorly, suggesting buparlisib plus fulvestrant may not be adequately effective against tumors with these poor prognostic molecular features. Clin Cancer Res; 22(7); 1583–91. ©2015 AACR.