ENPP1 is an innate immune checkpoint of the anticancer cGAMP-STING pathway in breast cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) expression correlates with poor prognosis in many cancers, and we previously discovered that ENPP1 is the dominant hydrolase of extracellular cGAMP: a cancer-cell-produced immunotransmitter that activates the anticancer stimulator of interferon genes (STING) pathway. However, ENPP1 has other catalytic activities and the molecular and cellular mechanisms contributing to its tumorigenic effects remain unclear. Here, using single-cell RNA-seq, we show that ENPP1 in both cancer and normal tissues drives primary breast tumor growth and metastasis by dampening extracellular 2'3'-cyclic-GMP-AMP (cGAMP)-STING-mediated antitumoral immunity. ENPP1 loss-of-function in both cancer cells and normal tissues slowed primary tumor growth and abolished metastasis. Selectively abolishing the cGAMP hydrolysis activity of ENPP1 phenocopied ENPP1 knockout in a STING-dependent manner, demonstrating that restoration of paracrine cGAMP-STING signaling is the dominant anti-cancer mechanism of ENPP1 inhibition. Finally, ENPP1 expression in breast tumors deterministically predicated whether patients would remain free of distant metastasis after pembrolizumab (anti-PD-1) treatment followed by surgery. Altogether, ENPP1 blockade represents a strategy to exploit cancer-produced extracellular cGAMP for controlled local activation of STING and is therefore a promising therapeutic approach against breast cancer.

Integrating Imaging and Circulating Tumor DNA Features for Predicting Patient Outcomes.

Biomarkers for evaluating tumor response to therapy and estimating the risk of disease relapse represent tremendous areas of clinical need. To evaluate treatment efficacy, tumor response is routinely assessed using different imaging modalities like positron emission tomography/computed tomography or magnetic resonance imaging. More recently, the development of circulating tumor DNA detection assays has provided a minimally invasive approach to evaluate tumor response and prognosis through a blood test (liquid biopsy). Integrating imaging- and circulating tumor DNA-based biomarkers may lead to improvements in the prediction of patient outcomes. For this mini-review, we searched the scientific literature to find original articles that combined quantitative imaging and circulating tumor DNA biomarkers to build prediction models. Seven studies reported building prognostic models to predict distant recurrence-free, progression-free, or overall survival. Three discussed building models to predict treatment response using tumor volume, pathologic complete response, or objective response as endpoints. The limited number of articles and the modest cohort sizes reported in these studies attest to the infancy of this field of study. Nonetheless, these studies demonstrate the feasibility of developing multivariable response-predictive and prognostic models using regression and machine learning approaches. Larger studies are warranted to facilitate the building of highly accurate response-predictive and prognostic models that are generalizable to other datasets and clinical settings.

Locoregional Breast Cancer Recurrence in the European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 MINDACT Trial: Analysis of Risk Factors Including the 70-Gene Signature.

PURPOSE: A number of studies are currently investigating de-escalation of radiation therapy in patients with a low risk of in-breast relapses on the basis of clinicopathologic factors and molecular tests. We evaluated whether 70-gene risk score is associated with risk of locoregional recurrence (LRR) and estimated 8-year cumulative incidences for LRR in patients with early-stage breast cancer treated with breast conservation. METHODS: In this exploratory substudy of European Organisation for Research and Treatment of Cancer 10041/BIG 03-04 MINDACT trial, we evaluated women with a known clinical and genomic 70-gene risk score test result and who had breast-conserving surgery (BCS). The primary end point was LRR at 8 years, estimated by cumulative incidences. Distant metastasis and death were considered competing risks. RESULTS: Among 6,693 enrolled patients, 5,470 (81.7%) underwent BCS, of whom 98% received radiotherapy. At 8-year follow-up, 189 patients experienced a LRR, resulting in an 8-year cumulative incidence of 3.2% (95% CI, 2.7 to 3.7). In patients with a low-risk 70-gene signature, the 8-year LRR incidence was 2.7% (95% CI, 2.1 to 3.3). In univariable analysis, adjusted for chemotherapy, five of 12 variables were associated with LRR, including the 70-gene signature. In multivariable modeling, adjuvant endocrine therapy and to a lesser extent tumor size and grade remained significantly associated with LRR. CONCLUSION: This exploratory analysis of the MINDACT trial estimated an 8-year low LRR rate of 3.2% after BCS. The 70-gene signature was not independently predictive of LRR perhaps because of the low number of events observed and currently cannot be used in clinical decision making regarding LRR. The overall low number of events does provide an opportunity to design trials toward de-escalation of local therapy.

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.

Prediction of Benefit From Adjuvant Pertuzumab by 80-Gene Signature in the APHINITY (BIG 4-11) Trial.

PURPOSE: At the primary analysis, the APHINITY trial reported a statistically significant but modest benefit of adding pertuzumab to standard adjuvant chemotherapy plus trastuzumab in patients with histologically confirmed human epidermal growth factor receptor 2 (HER2)-positive early-stage breast cancer. This study evaluated whether the 80-gene molecular subtyping signature (80-GS) could identify patients within the APHINITY population who derive the most benefit from dual anti-HER2 therapy. METHODS: In a nested case-control study design of 1,023 patients (matched event to control ratio of 3:1), the 80-GS classified breast tumors into functional luminal type, HER2 type, or basal type. Additionally, 80-GS distinguished tumor subtypes that exhibited a single-dominant functional pathway versus tumors with multiple activated pathways. The primary end point was invasive disease-free survival (IDFS). Hazard ratios (HRs) were evaluated by Cox regression. After excluding patients without appropriate consent and those with missing data, 964 patients were included. RESULTS: The 80-GS classified 50% (n = 479) of tumors as luminal type, 28% (n = 275) as HER2 type, and 22% (n = 209) as basal type. Most luminal-type tumors (86%) displayed a single-activated pathway, whereas 49% of HER2-type and 42% of basal-type tumors were dual activated. There was no significant difference in IDFS among different conventional 80-GS subtypes (single- and dual-activated subtypes combined). However, basal single-subtype tumors were significantly more likely to have an IDFS event (hazard ratio, 1.69 [95% CI, 1.12 to 2.54]) compared with other subtypes. HER2 single-subtype tumors displayed a trend toward greater beneficial effect on the addition of pertuzumab (hazard ratio, 0.56 [95% CI, 0.27 to 1.16]) compared with all other subtypes. CONCLUSION: The 80-GS identified subgroups of histologically confirmed HER2-positive tumors with distinct biological characteristics. Basal single-subtype tumors exhibit an inferior prognosis compared with other subgroups and may be candidates for additional therapeutic strategies. Preliminary results suggest patients with HER2-positive, genomically HER2 single-subtype tumors may particularly benefit from added pertuzumab, which warrants further investigation.

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.

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.

Utility of the 70-Gene MammaPrint Assay for Prediction of Benefit From Extended Letrozole Therapy in the NRG Oncology/NSABP B-42 Trial.

PURPOSE: MammaPrint (MP) determines distant metastatic risk and may improve patient selection for extended endocrine therapy (EET). This study examined MP in predicting extended letrozole therapy (ELT) benefit in patients with early-stage breast cancer (BC) from the NSABP B-42 trial. PATIENTS AND METHODS: MP was tested in 1,866 patients randomly assigned to receive ELT or placebo. The primary end point was distant recurrence (DR). Secondary end points were disease-free survival (DFS) and BC-free interval (BCFI). Tumors were classified as MP high risk (MP-HR) or low risk (MP-LR). MP-LR tumors were further classified as ultralow risk (MP-UL) or low non-ultralow risk (MP-LNUL). RESULTS: There was no statistically significant difference in ELT benefit on DR between MP-HR and MP-LR (interaction P = .38). MP-LR tumors (n = 1,160) exhibited a statistically significant 10-year benefit of 3.7% for DR (hazard ratio [HR], 0.43 [95% CI, 0.25 to 0.74]; P = .002), whereas MP-HR tumors (n = 706) exhibited a nonsignificant 2.4% benefit (HR, 0.65 [95% CI, 0.34 to 1.24]; P = .19). The 10-year ELT benefit was significant for DFS (7.8%) and BCFI (7.0%) for MP-LR tumors, whereas MP-HR tumors did not significantly benefit (interaction DFS: P = .015, BCFI: P = .006). In exploratory analysis, the 10-year ELT benefit was significant and more pronounced in MP-LNUL (n = 908) tumors: 4.0% for DR, 9.5% for DFS, and 7.9% for BCFI; the benefit in MP-UL (n = 252) tumors was not significant: 3% for DR, 1.8% for DFS, and 4.1% for BCFI. CONCLUSION: The primary hypothesis of predictive ability of MP on DR was not confirmed. However, the secondary outcomes demonstrated MP was predictive of ELT response and identified a subset of patients with early-stage hormone receptor-positive BC (MP-LR) with improved outcomes from ELT. These data could have important clinical implications in patient selection beyond clinical risk assessment for EET.

Race, Gene Expression Signatures, and Clinical Outcomes of Patients With High-Risk Early Breast Cancer.

Importance: There has been little consideration of genomic risk of recurrence by breast cancer subtype despite evidence of racial disparities in breast cancer outcomes. Objective: To evaluate associations between clinical trial end points, namely pathologic complete response (pCR) and distant recurrence-free survival (DRFS), and race and examine whether gene expression signatures are associated with outcomes by race. Design, Setting, and Participants: This retrospective cohort study used data from the Investigation of Serial Studies to Predict Your Therapeutic Response With Imaging and Molecular Analysis 2 (I-SPY 2) multicenter clinical trial of neoadjuvant chemotherapy with novel agents and combinations for patients with previously untreated stage II/III breast cancer. Analyses were conducted of associations between race and short- and long-term outcomes, overall and by receptor subtypes, and their association with 28 expression biomarkers. The trial enrolled 990 female patients between March 30, 2010, and November 5, 2016, with a primary tumor size of 2.5 cm or greater and clinical or molecular high risk based on MammaPrint or hormone receptor (HR)-negative/ERBB2 (formerly HER2 or HER2/neu)-positive subtyping across 9 arms. This data analysis was performed between June 10, 2021, and October 20, 2022. Exposure: Race, tumor receptor subtypes, and genomic biomarker expression of early breast cancer. Main Outcomes and Measures: The primary outcomes were pCR and DRFS assessed by race, overall, and by tumor subtype using logistic regression and Cox proportional hazards regression models. The interaction between 28 expression biomarkers and race, considering pCR and DRFS overall and within subtypes, was also evaluated. Results: The analytic sample included 974 participants (excluding 16 self-reporting as American Indian or Alaska Native, Native Hawaiian or Other Pacific Islander, or multiple races due to small sample sizes), including 68 Asian (7%), 120 Black (12%), and 786 White (81%) patients. Median (range) age at diagnosis was 47 (25-71) years for Asian, 49 (25-77) for Black, and 49 (23-73) years for White patients. The pCR rates were 32% (n = 22) for Asian, 30% for Black (n = 36), and 32% for White (n = 255) patients (P = .87). Black patients with HR-positive/ERBB2-negative tumors not achieving pCR had significantly worse DRFS than their White counterparts (hazard ratio, 2.28; 95% CI, 1.24-4.21; P = .01), with 5-year DRFS rates of 55% (n = 32) and 77% (n = 247), respectively. Black patients with HR-positive/ERBB2-negative tumors, compared with White patients, had higher expression of an interferon signature (mean [SD], 0.39 [0.87] and -0.10 [0.99]; P = .007) and, compared with Asian patients, had a higher mitotic score (mean [SD], 0.07 [1.08] and -0.69 [1.06]; P = .01) and lower estrogen receptor/progesterone receptor signature (mean [SD], 0.31 [0.90] and 1.08 [0.95]; P = .008). A transforming growth factor ß signature had a significant association with race relative to pCR and DRFS, with a higher signature associated with lower pCR and worse DRFS outcomes among Black patients only. Conclusions and Relevance: The findings show that women with early high-risk breast cancer who achieve pCR have similarly good outcomes regardless of race, but Black women with HR-positive/ERBB2-negative tumors without pCR may have worse DRFS than White women, highlighting the need to develop and test novel biomarker-informed therapies in diverse populations.