IMPORTANCE Patients with breast cancer (BrCa) brain metastases (BrM) have limited therapeutic options. A better understanding of molecular alterations acquired in BrM could identify clinically actionable metastatic dependencies. OBJECTIVE To determine whether there are intrinsic subtype differences between primary tumors and matched BrM and to uncover BrM-acquired alterations that are clinically actionable. DESIGN, SETTING AND PARTICPANTS In total, 20 cases of primary breast cancer tissue and resected BrM (10 estrogen receptor [ER]-negative and 10 ER-positive) from 2 academic institutions were included. Eligible cases in the discovery cohort harbored patient-matched primary breast cancer tissue and resected BrM. Given the rarity of patient-matched samples, no exclusion criteria were enacted. Two validation sequencing cohorts were used—a published data set of 17 patient-matched cases of BrM and a cohort of 7884 BrCa tumors enriched for metastatic samples. MAIN OUTCOMES AND MEASURES Brain metastases expression changes in 127 genes within BrCa signatures, PAM50 assignments, and ERBB2/HER2 DNA-level gains. RESULTS Overall, 17 of 20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17 of 20 BrM harbored expression changes (<2-fold or >2-fold) in clinically actionable genes including gains of FGFR4 (n = 6 [30%]), FLT1 (n = 4 [20%]), AURKA (n = 2 [10%]) and loss of ESR1 expression (n = 9 [45%]). The most recurrent expression gain was ERBB2/HER2, which showed a greater than 2-fold expression increase in 7 of 20 BrM (35%). Three of these 7 cases were ERBB2/HER2-negative out of 13 ERBB2/HER2-negative in the primary BrCa cohort and became immunohistochemical positive (3+) in the paired BrM with metastasis-specific amplification of the ERBB2/HER2 locus. In an independent data set, 2 of 9 (22.2%) ERBB2/HER2-negative BrCa switched to ERBB2/HER2-positive with 1 BrM acquiring ERBB2/HER2 amplification and the other showing metastatic enrichment of the activating V777L ERBB2/HER2 mutation. An expanded cohort revealed that ERBB2/HER2 amplification and/or mutation frequency was unchanged between local disease and metastases across all sites; however, a significant enrichment was appreciated for BrM (13% local vs 24% BrM; P < .001). CONCLUSIONS AND RELEVANCE Breast cancer BrM commonly acquire alterations in clinically actionable genes, with metastasis-acquired ERBB2/HER2 alterations in approximately 20% of ERBB2/HER2-negative cases. These observations have immediate clinical implications for patients with ERBB2/HER2–negative breast cancer and support comprehensive profiling of metastases to inform clinical care.

RNA-seq profiling of longitudinally collected specimens uncovered recurrent gene expression acquisitions in metastatic tumors, distinct from matched primary tumors. Critically, we identify aberrations in key oncogenic pathways and provide functional evidence for their suitability as therapeutic targets. Altogether, this study establishes recurrent, acquired vulnerabilities in BrM that warrant immediate clinical investigation and suggests paired specimen expression profiling as a compelling and underutilized strategy to identify targetable dependencies in advanced cancers.

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BackgroundInvasive lobular carcinoma (ILC) as a disease entity distinct from invasive ductal carcinoma (IDC) has merited focused studies of the genomic landscape, but those to date are largely limited to the assessment of early-stage cancers. Given that genomic alterations develop as acquired resistance to endocrine therapy, studies on refractory ILC are needed.Patients and methodsTissue from 336 primary-enriched, breast-biopsied ILC and 485 estrogen receptor (ER)-positive IDC and metastatic biopsy specimens from 180 ILC and 191 ER-positive IDC patients was assayed with hybrid-capture-based comprehensive genomic profiling for short variant, indel, copy number variants, and rearrangements in up to 395 cancer-related genes.ResultsWhereas ESR1 alterations are enriched in the metastases of both ILC and IDC compared with breast specimens, NF1 alterations are enriched only in ILC metastases (mILC). NF1 alterations are predominantly under loss of heterozygosity (11/14, 79%), are mutually exclusive with ESR1 mutations [odds ratio = 0.24, P < 0.027] and are frequently polyclonal in ctDNA assays. Assessment of paired specimens shows that NF1 alterations arise in the setting of acquired resistance. An in vitro model of CDH1 mutated ER-positive breast cancer demonstrates that NF1 knockdown confers a growth advantage in the presence of 4-hydroxy tamoxifen. Our study further identified a significant increase in tumor mutational burden (TMB) in mILCs relative to breast ILCs or metastatic IDCs (8.9% >20 mutations/mb; P < 0.001). Most TMB-high mILCs harbor an APOBEC trinucleotide signature (14/16; 88%).ConclusionsThis study identifies alteration of NF1 as enriched specifically in mILC. Mutual exclusivity with ESR1 alterations, polyclonality in relapsed ctDNA, and de novo acquisition suggest a role for NF1 loss in endocrine therapy resistance. Since NF1 loss leads to RAS/RAF kinase activation, patients may benefit from a matched inhibitor. Moreover, for an independent subset of mILC, TMB was elevated relative to breast ILC, suggesting possible benefit from immune checkpoint inhibitors.

Invasive lobular carcinoma (ILC) is an understudied subtype of breast cancer that requires novel therapies in the advanced setting. To study acquired resistance to endocrine therapy in ILC, we have recently performed RNA-Sequencing on long-term estrogen deprived cell lines and identified FGFR4 overexpression as a top druggable target. Here, we show that FGFR4 expression also increases dramatically in endocrine-treated distant metastases, with an average fold change of 4.8 relative to the paired primary breast tumor for ILC, and 2.4-fold for invasive ductal carcinoma (IDC). In addition, we now report that FGFR4 hotspot mutations are enriched in metastatic breast cancer, with an additional enrichment for ILC, suggesting a multimodal selection of FGFR4 activation. These data collectively support the notion that FGFR4 is an important mediator of endocrine resistance in ILC, warranting future mechanistic studies on downstream signaling of overexpressed wild-type and mutant FGFR4.

DNA sequencing has identified a limited number of driver mutations in metastatic breast cancer beyond single basepair mutations in the estrogen receptor (ESR1). However, our previous studies and others have observed that structural variants, such as ESR1 fusions, may also play a role. Therefore, we expanded upon these observations by performing a comprehensive and highly sensitive characterization of copy-number (CN) alterations in a large clinical cohort of metastatic specimens. NanoString DNA hybridization was utilized to measure CN gains, amplifications, and deletions of 67 genes in 108 breast cancer metastases, and in 26 cases, the patient-matched primary tumor. For ESR1, a copyshift algorithm was applied to identify CN imbalances at exon-specific resolution and queried large data sets (>15,000 tumors) that had previously undergone next-generation sequencing (NGS). Interestingly, a subset of ER þ tumors showed increased ESR1 CN (11/82, 13%); three had CN amplifications (4%) and eight had gains (10%). Increased ESR1 CN was enriched in metastatic specimens versus primary tumors, and this was orthogonally confirmed in a large NGS data set. ESR1-amplified tumors showed a site-specific enrichment for bone metastases and worse outcomes than nonamplified tumors. No ESR1 CN amplifications and only one gain was identified in ER À tumors. ESR1 copyshift was present in 5 of the 11 ESR1-amplified tumors. Other frequent amplifications included ERBB2, GRB7, and cell-cycle pathway members CCND1 and CDK4/6, which showed mutually exclusivity with deletions of CDKN2A, CDKN2B, and CDKN1B. Implications: Copy-number alterations of ESR1 and key CDK pathway genes are frequent in metastatic breast cancers, and their clinical relevance should be tested further.

Abstract Invasive lobular breast carcinoma (ILC) accounts for 10-15% of breast cancers diagnosed annually. Evidence suggests that some aspects of endocrine treatment response might differ between invasive ductal carcinoma (IDC) and ILC, and that patients with ILC have worse long-term survival. We analyzed The Cancer Genome Atlas dataset and observed lower levels of ESR1 mRNA (P=0.002) and ERα protein (P=0.038) in ER+ ILC (n=137) compared to IDC (n=554) and further confirmed the mRNA difference in a local UPMC cohort (ILC, n=143; IDC, n=877; P&lt;0.005). In both datasets, the correlation between ESR1 mRNA and ERα protein was weaker in ILC suggesting differential post-transcriptional regulation of ERα. In vitro, 17β-estradiol (E2) decreased the rate of degradation and increased the half-life of ERα in ILC cell lines, whereas the opposite was observed in IDC cell lines. Further, E2 failed to induce robust ubiquitination of ERα in ILC cells. To determine the potential clinical relevance of these findings, we evaluated the effect of two selective estrogen receptor down-regulators (SERDs), ICI182,780 and AZD9496, on ERα turnover and cell growth. While ICI182,780 and AZD9496 showed similar effects in IDC cells, in ILC cell lines, AZD9496 was not as effective as ICI182,780 in decreasing ERα stability and E2 induced proliferation. Furthermore, AZD9496 exhibited partial agonist activity in growth assays in ILC cell lines. Our study provides evidence for a distinct ERα regulation by SERDs in ILC cell lines and therefore it is important to include ILC models into preclinical and clinical testing of novel SERDs.

Over the years, immune checkpoint inhibitors (CPIs) have become a powerful treatment strategy in the field of cancer immunotherapy. In the last decade, the number of FDA-approved CPIs has been increasing prominently, opening new horizons for the treatment of a wide range of tumor types. Pointedly, three immune checkpoint molecules have been under extensive research, which include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1) and its ligand-1 (PD-L1). Despite remarkable success, not all patients respond positively to therapy, which highlights the complexity of the tumor microenvironment (TME) and immune system. This has led to the identification of molecular biomarkers to predict response and toxicity. In addition, there has been an emerging focus on developing new delivery and targeting approaches for better drug efficacy and potency. In this review, we highlight the mechanism of action of major CPIs, their clinical impact, variation in effectiveness, response prediction, updated clinical indications, current challenges and limitations, promising novel approaches, and future directions.