Differential Kinase Activity Across Prostate Tumor Compartments Defines Sensitivity to Target Inhibition.

Cancer therapy often results in heterogeneous responses in different metastatic lesions in the same patient. Inter- and intratumor heterogeneity in signaling within various tumor compartments and its impact on therapy are not well characterized due to the limited sensitivity of single-cell proteomic approaches. To overcome this barrier, we applied single-cell mass cytometry with a customized 26-antibody panel to PTEN-deleted orthotopic prostate cancer xenograft models to measure the evolution of kinase activities in different tumor compartments during metastasis or drug treatment. Compared with primary tumors and circulating tumor cells (CTC), bone metastases, but not lung and liver metastases, exhibited elevated PI3K/mTOR signaling and overexpressed receptor tyrosine kinases (RTK) including c-MET protein. Suppression of c-MET impaired tumor growth in the bone. Intratumoral heterogeneity within tumor compartments also arose from highly proliferative EpCAM-high epithelial cells with increased PI3K and mTOR kinase activities coexisting with poorly proliferating EpCAM-low mesenchymal populations with reduced kinase activities; these findings were recapitulated in epithelial and mesenchymal CTC populations in patients with metastatic prostate and breast cancer. Increased kinase activity in EpCAM-high cells rendered them more sensitive to PI3K/mTOR inhibition, and drug-resistant EpCAM-low populations with reduced kinase activity emerged over time. Taken together, single-cell proteomics indicate that microenvironment- and cell state-dependent activation of kinase networks create heterogeneity and differential drug sensitivity among and within tumor populations across different sites, defining a new paradigm of drug responses to kinase inhibitors. SIGNIFICANCE: Single-cell mass cytometry analyses provide insights into the differences in kinase activities across tumor compartments and cell states, which contribute to heterogeneous responses to targeted therapies.

NR4A1 regulates expression of immediate early genes, suppressing replication stress in cancer.

Deregulation of oncogenic signals in cancer triggers replication stress. Immediate early genes (IEGs) are rapidly and transiently expressed following stressful signals, contributing to an integrated response. Here, we find that the orphan nuclear receptor NR4A1 localizes across the gene body and 3' UTR of IEGs, where it inhibits transcriptional elongation by RNA Pol II, generating R-loops and accessible chromatin domains. Acute replication stress causes immediate dissociation of NR4A1 and a burst of transcriptionally poised IEG expression. Ectopic expression of NR4A1 enhances tumorigenesis by breast cancer cells, while its deletion leads to massive chromosomal instability and proliferative failure, driven by deregulated expression of its IEG target, FOS. Approximately half of breast and other primary cancers exhibit accessible chromatin domains at IEG gene bodies, consistent with this stress-regulatory pathway. Cancers that have retained this mechanism in adapting to oncogenic replication stress may be dependent on NR4A1 for their proliferation.

A Digital RNA Signature of Circulating Tumor Cells Predicting Early Therapeutic Response in Localized and Metastatic Breast Cancer.

The multiplicity of new therapies for breast cancer presents a challenge for treatment selection. We describe a 17-gene digital signature of breast circulating tumor cell (CTC)-derived transcripts enriched from blood, enabling high-sensitivity early monitoring of response. In a prospective cohort of localized breast cancer, an elevated CTC score after three cycles of neoadjuvant therapy is associated with residual disease at surgery (P = 0.047). In a second prospective cohort with metastatic breast cancer, baseline CTC score correlates with overall survival (P = 0.02), as does persistent CTC signal after 4 weeks of treatment (P = 0.01). In the subset with estrogen receptor (ER)-positive disease, failure to suppress ER signaling within CTCs after 3 weeks of endocrine therapy predicts early progression (P = 0.008). Drug-refractory ER signaling within CTCs overlaps partially with presence of ESR1 mutations, pointing to diverse mechanisms of acquired endocrine drug resistance. Thus, CTC-derived digital RNA signatures enable noninvasive pharmacodynamic measurements to inform therapy in breast cancer.Significance: Digital analysis of RNA from CTCs interrogates treatment responses of both localized and metastatic breast cancer. Quantifying CTC-derived ER signaling during treatment identifies patients failing to respond to ER suppression despite having functional ESR1. Thus, noninvasive scoring of CTC-RNA signatures may help guide therapeutic choices in localized and advanced breast cancer. Cancer Discov; 8(10); 1286-99. ©2018 AACR. This article is highlighted in the In This Issue feature, p. 1195.

Molecular signatures of circulating melanoma cells for monitoring early response to immune checkpoint therapy.

A subset of patients with metastatic melanoma have sustained remissions following treatment with immune checkpoint inhibitors. However, analyses of pretreatment tumor biopsies for markers predictive of response, including PD-1 ligand (PD-L1) expression and mutational burden, are insufficiently precise to guide treatment selection, and clinical radiographic evidence of response on therapy may be delayed, leading to some patients receiving potentially ineffective but toxic therapy. Here, we developed a molecular signature of melanoma circulating tumor cells (CTCs) to quantify early tumor response using blood-based monitoring. A quantitative 19-gene digital RNA signature (CTC score) applied to microfluidically enriched CTCs robustly distinguishes melanoma cells, within a background of blood cells in reconstituted and in patient-derived (n = 42) blood specimens. In a prospective cohort of 49 patients treated with immune checkpoint inhibitors, a decrease in CTC score within 7 weeks of therapy correlates with marked improvement in progression-free survival [hazard ratio (HR), 0.17; P = 0.008] and overall survival (HR, 0.12; P = 0.04). Thus, digital quantitation of melanoma CTC-derived transcripts enables serial noninvasive monitoring of tumor burden, supporting the rational application of immune checkpoint inhibition therapies.

An RNA-Based Digital Circulating Tumor Cell Signature Is Predictive of Drug Response and Early Dissemination in Prostate Cancer.

Blood-based biomarkers are critical in metastatic prostate cancer, where characteristic bone metastases are not readily sampled, and they may enable risk stratification in localized disease. We established a sensitive and high-throughput strategy for analyzing prostate circulating tumor cells (CTC) using microfluidic cell enrichment followed by digital quantitation of prostate-derived transcripts. In a prospective study of 27 patients with metastatic castration-resistant prostate cancer treated with first-line abiraterone, pretreatment elevation of the digital CTCM score identifies a high-risk population with poor overall survival (HR = 6.0; P = 0.01) and short radiographic progression-free survival (HR = 3.2; P = 0.046). Expression of HOXB13 in CTCs identifies 6 of 6 patients with ≤12-month survival, with a subset also expressing the ARV7 splice variant. In a second cohort of 34 men with localized prostate cancer, an elevated preoperative CTCL score predicts microscopic dissemination to seminal vesicles and/or lymph nodes (P < 0.001). Thus, digital quantitation of CTC-specific transcripts enables noninvasive monitoring that may guide treatment selection in both metastatic and localized prostate cancer.Significance: There is an unmet need for biomarkers to guide prostate cancer therapies, for curative treatment of localized cancer and for application of molecularly targeted agents in metastatic disease. Digital quantitation of prostate CTC-derived transcripts in blood specimens is predictive of abiraterone response in metastatic cancer and of early dissemination in localized cancer. Cancer Discov; 8(3); 288-303. ©2018 AACR.See related commentary by Heitzer and Speicher, p. 269This article is highlighted in the In This Issue feature, p. 253.

An RNA-based signature enables high specificity detection of circulating tumor cells in hepatocellular carcinoma.

Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.