Transcriptome Characterization of Matched Primary Breast and Brain Metastatic Tumors to Detect Novel Actionable Targets

Abstract

Background: Breast cancer brain metastases (BrMs) are defined by complex adaptations to both adjuvant treatment regimens and the brain microenvironment. Consequences of these alterations remain poorly understood, as does their potential for clinical targeting. We utilized genome-wide molecular profiling to identify therapeutic targets acquired in metastatic disease.

Methods: Gene expression profiling of 21 patient-matched primary breast tumors and their associated brain metastases was performed by TrueSeq RNA-sequencing to determine clinically actionable BrM target genes. Identified targets were functionally validated using small molecule inhibitors in a cohort of resected BrM ex vivo explants (n = 4) and in a patient-derived xenograft (PDX) model of BrM. All statistical tests were two-sided.

Results: Considerable shifts in breast cancer cell-specific gene expression profiles were observed (1314 genes upregulated in BrM; 1702 genes downregulated in BrM; DESeq; fold change > 1.5, Padj < .05). Subsequent bioinformatic analysis for readily druggable targets revealed recurrent gains in RET expression and human epidermal growth factor receptor 2 (HER2) signaling. Small molecule inhibition of RET and HER2 in ex vivo patient BrM models (n = 4) resulted in statistically significantly reduced proliferation (P < .001 in four of four models). Furthermore, RET and HER2 inhibition in a PDX model of BrM led to a statistically significant antitumor response vs control (n = 4, % tumor growth inhibition [mean difference; SD], anti-RET = 86.3% [1176; 258.3], P < .001; anti-HER2 = 91.2% [1114; 257.9], P < .01).

Conclusions: 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.

Figure 1.

Transcriptome evolution in breast cancer brain metastasis. A) Correspondence analysis showing overall trends in paired samples (n = 21) using the gene expression of all protein coding genes. The matched primary (circles) and metastasis samples (squares) are paired via a connecting line. The first component (“Comp1”) represents the strongest trend and splits the samples from the primary to the metastasis; the other two components split the samples by intrinsic subtype. B) Unsupervised hierarchical clustering heatmap. Patient-matched primary and metastatic tumor samples (n = 21) that clustered as related pairs in the dendogram are indicated with an asterisk. C) PAM50 intrinsic molecular subtype calls in patient-matched samples (n = 21). Probability for each subtype is the mean of all 20-fold test probabilities; tile plot denotes this probability for each subtype. Diamonds indicate brain metastases with greater than 10% probability gain in PAM50 subtypes. Legend denotes PAM50 subtype (blue = luminal A, purple = luminal B, green = HER2, red = basal), hormone status (green = positive, black = negative), tissue source (yellow = Royal College of Surgeons, Ireland; purple = University of Pittsburgh, United States), and tumor site (blue = primary, red = metastasis). D) Recurrent differentially upregulated genes (n = 1314) were screened in two merged public metastatic cohorts (GSE14017/18). Heatmap displays 62 genes whose expression was upregulated in brain metastases (BrMs) but not in metastases to lung, liver, or bone (BrM-related gene set). E) Kaplan-Meier curves for brain metastasis–free survival of BrM-related gene set status in two cohorts (n = 268; GSE12276/2034). P value based on two-sided log-rank test. F) Schematic of the workflow for uncovering decontaminated brain metastasis–related genes. G) Kaplan-Meier curves for brain metastasis–free survival on the basis of decontaminated BrM-related gene set (n = 11) status in two cohorts (n = 268; GSE12276/2034). P value based on two-sided log-rank test. H) Gene set variation analysis utilizing MsigDB Oncogenic Pathway (MsigDB). Heatmap illustrates brain metastasis–enriched pathways (false discovery rate–adjusted Wilcoxon signed-ranked P < .05) in brain metastases vs primaries. BrM = brain metastasis; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; RCSI = Royal College of Surgeons in Ireland.

Figure 2.

Recurrent expression gains of clinically actionable kinase pathways in breast cancer brain metastases. A) Paired ladder plot of established genes represented in the human epidermal growth factor receptor 2 (HER2) signature depicts the expression change in patient-matched cases (P = .008; two-sided Wilcoxon signed-rank test; primaries vs brain metastases). Blue dots represent primary tumor signature scores, and red dots represent metastatic tumor signature scores. B) Scatter plot of HER2 signature score in primary tumors. Blue dots (-/-) represent patient-matched cases that are HER2 negative in both the primary and metastatic tumors, red dots (-/+) represent patient-matched cases that switched from HER2 negative to positive, whereas green dots (+/+) represent HER2-positive tumors that have further activation in the HER2 pathway. C) Tile plot indicates gain of HER2 signature or loss of ESR1 expression. Squares represent patient-matched cases that switched from HER2 negative to positive, whereas circles represent HER2-positive tumors that have further activation in HER2 pathways. D) Primary and metastatic log2normCPM values of ESR1/ERBB2 from case 4_RCS, along with immunohistochemistry protein analysis. Images shown are 20×; scale bars correspond to 50 μm. E) ESR1 gene differentially methylated regions (DMRs) identified with methyl capture sequencing are illustrated and were identified by comparing 4_RCS case primary and brain metastasis. Plot shows regions of hypermethylation and hypomethylation found in the ESR1 gene. F) OncoPrint of clinically actionable kinases (DGIdb) with discrete expression gains in brain metastases. G) Paired ladder plot of RET expression in patient-matched cases. Light green dots represent primary tumor expression values, and dark green dots represent metastatic tumor expression values (log2normCPM). Representative primary and metastatic immunohistochemistry (IHC) staining of RET protein from case 6_RCS, 72_PITT and 5_RCS; along with a graphic displaying RET IHC scores alongside corresponding log2FC RET mRNA scores for the sequenced paired samples. Images shown are 20×; scale bars correspond to 50 μm. BrM = brain metastasis; HER2 = human epidermal growth factor receptor 2; IHC = immunohistochemistry.

Figure 3.

Inhibition of RET and human epidermal growth factor receptor 2 (HER2) in breast cancer brain metastases ex vivo. A) Schematic of the ex vivo experimental set up. B) Immunohistochemistry (IHC) protein analysis of HER2/RET from case T638P (primary breast) and patient-matched T638 brain metastasis (BrM). Images shown are 20×; scale bars correspond to 50 μm. Also shown is mRNA expression levels of RET and key modules of HER2 signature (HER2, PSMD3, CASC3, GRB7, and N1RD1) analyzed by Taqman polymerase chain reaction. The bar chart displays ΔΔCt values for each gene. C) Brain metastatic tissue (x-BrMT606, T347, T638, and T681) was treated with vehicle (0.1% DMSO), 10 nM cabozantinib, and 25 nM afatinib and processed as described. IHC was carried out to profile ER, HER2, and RET of the ex vivo sample. Magnetic resonance/computed tomography images of the brain metastases resected are shown. Estrogen receptor, progesterone receptor, and HER2 status in primary and brain metastases are indicated alongside adjuvant treatment received before resection. Representative images of IHC analyses of Ki67 tumors treated for 72 hours with indicated treatments (positive cells indicated with red triangles). All analyses of variance, followed by Dunnett’s test. All statistical tests were two-sided. AC = cyclophosphamide/doxorubicin; AFA = afatinib; AI = aromatase inhibitor; BrM = brain metastasis; CABO = cabozantinib; ER = estrogen receptor; HER2 = human epidermal growth factor receptor 2; PR = progesterone receptor; qPCR = quantitative polymerase chain reaction; TAM = tamoxifen; T = taxol; TC = taxol/carboplatin; UCH = unknown chemotherapy; xBrM = brain metastases explant; XRT = radiotherapy; ZOM = zometa.

Figure 4.

Ex vivo effects of RET and human epidermal growth factor receptor 2 (HER2) inhibition on downstream signaling pathways. A) Analysis of cabozantinib antitumor efficacy in xBrM explants. xBrM explants were designated an RET immunohistochemistry (IHC) positivity score. The cabozantinib efficacy with respect to RET IHC score was calculated based on %ki67 inhibition where (100%- (mean ki67% cabozantinib-treated samples/mean ki67% DMSO-treated)*100 = %ki67 inhibition with cabozantinib treatment. xBrM T606 was analyzed for pRET(Y1062) IHC expression in vehicle (DMSO; V)- and cabozantinib (CABO)-treated samples. Representative images of IHC analyses of the tumors treated for 72 hours with indicated treatments. All scale bars = 50 μm. Error bars represent mean (SD) (n = 5–10 images per group), two-tailed paired t test. B) Afatinib efficacy with respect to ERBB2 amplification status and HER2 and pHER2 IHC score. C) Representative IHC staining of HER family members alongside key phosphorylated proteins pEGFR (Y¹⁰⁶⁸), pHER2 (Y¹²²¹), pHER3 (Y¹²⁸⁹), and pHER4 (Y¹²⁸⁴), along with a graphic displaying IHC scores for each of the brain metastatic tissues utilized in the study. Images shown are 20×; scale bars correspond to 50 μm. D) T606 was analyzed for pHER2 (Y¹²²¹) and pEGFR (Y¹⁰⁶⁸) IHC expression in vehicle (V)- and afatinib (AFA)-treated samples. Representative images of IHC analyses of the tumors treated for 72 hours with indicated treatments. All scale bars = 50 μm. Error bars represent mean (SD) (n = 5–10 images per group), two-tailed paired t test. AFA = afatinib; BrM = brain metastasis; CABO = cabozantinib; EGFR = epidermal growth factor receptor; HER2 = human epidermal growth factor receptor 2; IHC = immunohistochemistry; V = vehicle.

Figure 5.

Inhibition of RET and human epidermal growth factor receptor 2 (HER2) in breast cancer brain metastases in vivo. A) Schematic indicates clinical information pertaining to brain metastasis (BrM) patient-derived xenograft (PDX) CTG-1520 and the experimental design of the in vivo experiment. Representative immunohistochemistry (IHC) images of hematoxylin and eosin, pan cytokeratin, HER2, RET, pRET(Y1062), pEGFR (Y¹⁰⁶⁸), pHER3 (Y¹²⁸⁹), and pHER4 (Y¹²⁸⁴) are shown. Scale bars = 50 μm. B) Schematic of the in vivo experimental setup. Treatment schedule was four cycles (QD×5 on/2 off) via oral gavage of vehicle (black line), 30 mg/kg cabozantinib (red line), and 20 mg/kg afatinib (blue line). Effects on tumor growth were evaluated with % tumor growth inhibition (%TGI). The tumor growth curve shows mean tumor volume +/-SD (n = 4 per treatment group). The analysis of variance test was followed by Newman-Keuls multiple comparison test. All statistical tests were two-sided. C) CTG1520 PDX was analyzed for pRET(Y1062) IHC expression in vehicle (V)- and cabozantinib (CABO)-treated samples. Representative images of IHC analyses of the tumors treated analyzed at the conclusion of the experiment. All scale bars = 100 μm. Error bars represent mean ±SD (n = 5–10 images per group), two-sided paired t test. D) CTG1520 PDX was analyzed for pEGFR (Y¹⁰⁶⁸), pHER4 (Y¹²⁸⁴), and pERK (T²⁰²/Y²⁰⁴) IHC expression in vehicle (V)- and afatinib (AFA)-treated samples. Representative images of IHC analyses of the tumors analyzed at the conclusion of the experiment. All scale bars = 50 μm. Error bars represent mean (SD) (n = 5–10 images per group), two-tailed paired t test. AFA = afatinib; BrM = brain metastasis; CABO = cabozantinib; CMF = cyclophosphamide, methotrexate, 5-fluorouracil; DC = docetaxel/carboplatin; EGFR = epidermal growth factor receptor; FEC = fluorouracil (5FU), epirubicin, cyclophosphamide; H&E = hematoxylin and eosin; HER2 = human epidermal growth factor receptor 2; PTX = paclitaxel; V = vehicle.