Systematic annotation of orphan RNAs reveals blood-accessible molecular barcodes of cancer identity and cancer-emergent oncogenic drivers.

From extrachromosomal DNA to neo-peptides, the broad reprogramming of the cancer genome leads to the emergence of molecules that are specific to the cancer state. We recently described orphan non-coding RNAs (oncRNAs) as a class of cancer-specific small RNAs with the potential to play functional roles in breast cancer progression1. Here, we report a systematic and comprehensive search to identify, annotate, and characterize cancer-emergent oncRNAs across 32 tumor types. We also leverage large-scale in vivo genetic screens in xenografted mice to functionally identify driver oncRNAs in multiple tumor types. We have not only discovered a large repertoire of oncRNAs, but also found that their presence and absence represent a digital molecular barcode that faithfully captures the types and subtypes of cancer. Importantly, we discovered that this molecular barcode is partially accessible from the cell-free space as some oncRNAs are secreted by cancer cells. In a large retrospective study across 192 breast cancer patients, we showed that oncRNAs can be reliably detected in the blood and that changes in the cell-free oncRNA burden captures both short-term and long-term clinical outcomes upon completion of a neoadjuvant chemotherapy regimen. Together, our findings establish oncRNAs as an emergent class of cancer-specific non-coding RNAs with potential roles in tumor progression and clinical utility in liquid biopsies and disease monitoring.

Cell-free DNA Concentration as a Biomarker of Response and Recurrence in HER2-Negative Breast Cancer Receiving Neoadjuvant Chemotherapy.

PURPOSE: We previously demonstrated the clinical significance of circulating tumor DNA (ctDNA) in patients with HER2-negative breast cancer receiving neoadjuvant chemotherapy (NAC). Here, we compared its predictive and prognostic value with cell-free DNA (cfDNA) concentration measured in the same samples from the same patients. EXPERIMENTAL DESIGN: 145 patients with hormone receptor (HR)-positive/HER2-negative and 138 triple-negative breast cancer (TNBC) with ctDNA data from a previous study were included in the analysis. Associations of serial cfDNA concentration with residual cancer burden (RCB) and distant recurrence-free survival (DRFS) were examined. RESULTS: In TNBC, we observed a modest negative correlation between cfDNA concentration 3 weeks after treatment initiation and RCB, but none of the other timepoints showed significant correlation. In contrast, ctDNA was significantly positively correlated with RCB at all timepoints (all R > 0.3 and P < 0.05). In the HR-positive/HER2-negative group, cfDNA concentration did not associate with response to NAC, but survival analysis showed that high cfDNA shedders at pretreatment had a significantly worse DRFS than low shedders (hazard ratio, 2.12; P = 0.037). In TNBC, the difference in survival between high versus low cfDNA shedders at all timepoints was not statistically significant. In contrast, as previously reported, ctDNA at all timepoints was significantly correlated with DRFS in both subtypes. CONCLUSIONS: In TNBC, cfDNA concentrations during therapy were not strongly correlated with response or prognosis. In the HR-positive/HER2-negative group, pretreatment cfDNA concentration was prognostic for DRFS. Overall, the predictive and prognostic value of cfDNA concentration was more limited than that of ctDNA.

Epithelial Expressed B7-H4 Drives Differential Immunotherapy Response in Murine and Human Breast Cancer.

Combinations of immune checkpoint inhibitors (ICI, including anti-PD-1/PD-L1) and chemotherapy have been FDA approved for metastatic and early-stage triple-negative breast cancer (TNBC), but most patients do not benefit. B7-H4 is a B7 family ligand with proposed immunosuppressive functions being explored as a cancer immunotherapy target and may be associated with anti-PD-L1 resistance. However, little is known about its regulation and effect on immune cell function in breast cancers. We assessed murine and human breast cancer cells to identify regulation mechanisms of B7-H4 in vitro. We used an immunocompetent anti-PD-L1-sensitive orthotopic mammary cancer model and induced ectopic expression of B7-H4. We assessed therapy response and transcriptional changes at baseline and under treatment with anti-PD-L1. We observed B7-H4 was highly associated with epithelial cell status and transcription factors and found to be regulated by PI3K activity. EMT6 tumors with cell-surface B7-H4 expression were more resistant to immunotherapy. In addition, tumor-infiltrating immune cells had reduced immune activation signaling based on transcriptomic analysis. Paradoxically, in human breast cancer, B7-H4 expression was associated with survival benefit for patients with metastatic TNBC treated with carboplatin plus anti-PD-L1 and was associated with no change in response or survival for patients with early breast cancer receiving chemotherapy plus anti-PD-1. While B7-H4 induces tumor resistance to anti-PD-L1 in murine models, there are alternative mechanisms of signaling and function in human cancers. In addition, the strong correlation of B7-H4 to epithelial cell markers suggests a potential regulatory mechanism of B7-H4 independent of PD-L1. SIGNIFICANCE: This translational study confirms the association of B7-H4 expression with a cold immune microenvironment in breast cancer and offers preclinical studies demonstrating a potential role for B7-H4 in suppressing response to checkpoint therapy. However, analysis of two clinical trials with checkpoint inhibitors in the early and metastatic settings argue against B7-H4 as being a mechanism of clinical resistance to checkpoints, with clear implications for its candidacy as a therapeutic target.

Neoadjuvant Trebananib plus Paclitaxel-based Chemotherapy for Stage II/III Breast Cancer in the Adaptively Randomized I-SPY2 Trial-Efficacy and Biomarker Discovery.

PURPOSE: The neutralizing peptibody trebananib prevents angiopoietin-1 and angiopoietin-2 from binding with Tie2 receptors, inhibiting angiogenesis and proliferation. Trebananib was combined with paclitaxel±trastuzumab in the I-SPY2 breast cancer trial. PATIENTS AND METHODS: I-SPY2, a phase II neoadjuvant trial, adaptively randomizes patients with high-risk, early-stage breast cancer to one of several experimental therapies or control based on receptor subtypes as defined by hormone receptor (HR) and HER2 status and MammaPrint risk (MP1, MP2). The primary endpoint is pathologic complete response (pCR). A therapy "graduates" if/when it achieves 85% Bayesian probability of success in a phase III trial within a given subtype. Patients received weekly paclitaxel (plus trastuzumab if HER2-positive) without (control) or with weekly intravenous trebananib, followed by doxorubicin/cyclophosphamide and surgery. Pathway-specific biomarkers were assessed for response prediction. RESULTS: There were 134 participants randomized to trebananib and 133 to control. Although trebananib did not graduate in any signature [phase III probabilities: Hazard ratio (HR)-negative (78%), HR-negative/HER2-positive (74%), HR-negative/HER2-negative (77%), and MP2 (79%)], it demonstrated high probability of superior pCR rates over control (92%-99%) among these subtypes. Trebananib improved 3-year event-free survival (HR 0.67), with no significant increase in adverse events. Activation levels of the Tie2 receptor and downstream signaling partners predicted trebananib response in HER2-positive disease; high expression of a CD8 T-cell gene signature predicted response in HR-negative/HER2-negative disease. CONCLUSIONS: The angiopoietin (Ang)/Tie2 axis inhibitor trebananib combined with standard neoadjuvant therapy increased estimated pCR rates across HR-negative and MP2 subtypes, with probabilities of superiority >90%. Further study of Ang/Tie2 receptor axis inhibitors in validated, biomarker-predicted sensitive subtypes is warranted.