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Both transcriptomics and metabolomics hold promise for identifying acute coronary syndrome (ACS) but they have not been used in combination, nor have dynamic changes in levels been assessed as a diagnostic tool. We assessed integrated analysis of peripheral blood miRNA and metabolite analytes to distinguish patients with myocardial ischemia on cardiac stress testing. We isolated and quantified miRNA and metabolites before and after stress testing from seven patients with myocardial ischemia and 1:1 matched controls. The combined miRNA and metabolomic data were analyzed jointly in a supervised, dimension-reducing discriminant analysis. We implemented a baseline model (T0) and a stress-delta model. This novel integrative analysis of the baseline levels of metabolites and miRNA expression showed modest performance for distinguishing cases from controls. The stress-delta model showed worse performance. This pilot study shows potential for an integrated precision medicine approach to cardiac stress testing.The study of small sequences of ribonucleic acids (miRNAs) and byproducts of cellular metabolism (metabolites) could help us to identify important cardiac conditions such as not enough blood and oxygen supply to the heart (acute coronary syndrome). We obtained blood samples from patients getting cardiac stress tests (a noninvasive test to see if the patient has enough blood flow to their heart) before and after their test, then compared the levels of miRNAs and metabolites in them. We compared the levels in patients who had abnormal stress tests with those that had normal tests. We believe this could be a model for a new type of cardiac stress test if validated in more patients.

The change from baseline to C2D1 and C3D1 for each biomarker was determined across all patients. Biomarker change was statistically analyzed using Wilcoxon signed rank tests and the corresponding p-values, as well as the direction of change, are presented. As shown, Ang-2 levels decreased from baseline to C2D1 and baseline to C3D1; these changes were found to be statistically significant, p < 0.0001. Also, the directionality of change noted for Ang-2 at C2D1 and C3D1 was in the same direction (i.e., down). It should be noted that all biomarkers evaluated exhibited the same directionality of change at both C2D1 and C3D1. While many biomarkers exhibited consistent change at both time points, some biomarkers only exhibited statistically significant change at the C2D1 timepoint (i.e., D-dimer).

Regorafenib is a tyrosine kinase inhibitor approved by the FDA for the treatment of patients with chemotherapy refractory metastatic colorectal cancer (mCRC). Regorafenib inhibits signaling through multiple receptors associated with angiogenesis, metastasis, and tumor immunity. Here, we report biomarker results from LCCC1029, a randomized, placebo-controlled, phase II trial of chemotherapy ± regorafenib in patients with second-line mCRC. A panel of 20 soluble protein biomarkers (termed the Angiome) was assessed in the plasma of 149 patients from the LCCC1029 trial both at baseline and along the treatment continuum. Baseline protein levels were analyzed for prognostic and predictive value for progression-free survival (PFS) and overall survival (OS). Changes in protein levels during treatment were analyzed for potential pharmacodynamic effects. Six markers (HGF, IL6, PlGF, VEGF-R1, OPN, and IL6R) were found to be prognostic for PFS. Nine markers (IL6, TIMP-1, PlGF, VCAM-1, ICAM-1, OPN, TSP-2, HGF, and VEGF-R1) were prognostic for OS. Higher baseline levels of OPN (Pintx = 0.0167), VCAM-1 (Pintx = 0.0216), and PDGF-AA (Pintx = 0.0435) appeared to predict for PFS benefit from regorafenib compared with placebo. VCAM-1 was also potentially predictive of OS benefit from regorafenib compared with placebo (Pintx = 0.0124). On-treatment changes of six markers reflected potential on-target effect of regorafenib. Consistent results were observed in an Italian cohort where 105 patients with late-stage mCRC received regorafenib monotherapy. The key findings of this study suggest that VCAM-1 may be a predictive biomarker for regorafenib benefit, while multiple protein markers may be prognostic of outcome in patients with mCRC.

Hierarchical clustering analyses may identify potential patterns of marker expression and biologically relevant groupings. As an example, PDGF-AA and PDGF-BB are tightly clustered at baseline, indicating that if a patient had high PDGF-AA levels, PDGF-BB levels also tended to be high. This relationship is maintained at later time points (C2D1 and C3D1), as the clustering of PDGFs reflects similar patterns of change on-treatment.

A novel combination of bevacizumab and everolimus was evaluated in refractory colorectal cancer patients in a phase II trial. In this retrospective analysis, plasma samples from 49 patients were tested for over 40 biomarkers at baseline and after one or two cycles of drug administration. Analyte levels at baseline and change on-treatment were correlated with progression-free survival (PFS) and overall survival (OS) using univariate Cox proportional hazard modeling. Multivariable analyses were conducted using Cox modeling. Significant changes in multiple markers were observed following bevacizumab and everolimus treatment. Baseline levels of six markers significantly correlated with PFS and OS, including CRP, Gro-α, IGFBP-1, TF, ICAM-1, and TSP-2 (P < 0.05). At C2D1, changes of IGFBP-3, TGFβ-R3, and IGFBP-2 correlated with PFS and OS. Prognostic models were developed for OS and PFS (P = 0.0002 and 0.004, respectively). The baseline model for OS consisted of CRP, Gro-α, and TF, while the on-treatment model at C2D1 included IGFBP-2, IGFBP-3, and TGFβ-R3. These data demonstrated that multiple biomarkers were significantly modulated in response to bevacizumab and everolimus. Several markers correlated with both PFS and OS. Interestingly, these markers are known to be associated with inflammation and IGF signaling, key modulators of mTOR biology. Mol Cancer Ther; 14(4); 1048–56. ©2015 AACR.

Dendrograms

Biomarkers change on treatment

Figure S1, consort diagram. Table S1, pts characteristics. Table S2, baseline levels. Table S3, prognostic markers. Table S4, adjusted prognostic markers. Table S5, early changes. Table S6, late changes at disease progression.

TRC105 is an anti-endoglin antibody currently being tested in combination with VEGF inhibitors. In the phase Ib trial, 38 patients were treated with both TRC105 and bevacizumab (BEV), and improved clinical outcomes were observed, despite the fact that 30 patients (79%) were refractory to prior anti-VEGF therapy. Plasma samples were tested for angiogenic and inflammatory biomarkers at baseline and on-treatment. To provide broader context of this combination biomarker study, direct cross-study comparisons were made to biomarker studies previously conducted in patients treated with either BEV or TRC105 monotherapy. Upon treatment with BEV and TRC105, pharmacodynamic changes in response to both BEV (PlGF increase) and TRC105 (soluble endoglin increase) were noted. In addition, distinct patterns of change were identified (similar, opposing, neutralizing). Similar patterns were observed when the combination elicited similar effects to those observed with monotherapy treatment (i.e., decreases of Ang-2, increases of IL6 and VCAM-1). Opposing patterns were observed when the combination led to opposing effects compared with monotherapy treatment (i.e., TGFβ1, PDGF-AA and PDGF-BB, PAI-1). Lastly, neutralizing patterns were observed when one drug led to increase, whereas the other drug led to decrease, and the combination elicited no overall effect on the marker (i.e., VEGF-A, VEGF-D, and IGFBP-3). Patients achieving partial responses or stable disease from the combination exhibited significantly lower expression of E-Cadherin, HGF, ICAM-1, and TSP-2 at baseline. Taken together, the novel biomarker modulations identified may deepen our understanding of the underlying biology in patients treated with BEV and TRC105 compared with either drug alone. Mol Cancer Ther; 17(10); 2248–56. ©2018 AACR.

Supplementary Data from Predictive Biomarkers of Overall Survival in Patients with Metastatic Renal Cell Carcinoma Treated with IFNα ± Bevacizumab: Results from CALGB 90206 (Alliance)

All supplementary data

Supplemental Table 2. BEE study patients and the number of patients whose granulation tissue biopsies were available for biomarker study.

Supplementary Tables 1-2, Figures 1-4. Supplementary Table 1: List of assay primer sets used in this study. Supplementary Table 2. Table of co-regulation among the 14 genes using a Kendall tau analysis. Supplementary Figure 1: Forest plots showing associations of gene expression levels with OS in KRAS WT (A) and KRAS Mut (B) pts. All assayed genes are shown. Supplementary Figure 2: Forest plots showing associations of gene expression levels with PFS in KRAS WT (A) and KRAS Mut (B) pts. Supplementary Figure 3: Forest plots showing associations of gene expression levels and treatment group with OS in KRAS WT (A) and KRAS Mut (B) pts. Supplementary Figure 4: Forest plots showing associations of gene expression levels and treatment group with PFS in KRAS WT (A) and KRAS Mut (B) pts. All assayed genes are shown.

Supplementary methods, tables, figures 1-5 with corresponding figure legends, and references

Purpose:Endoglin is a coreceptor for TGFβ ligands that is highly expressed on proliferating endothelial cells and other cells in the tumor microenvironment. Clinical studies have noted increased programmed cell death (PD)-1 expression on cytotoxic T cells in the peripheral blood of patients with cancer treated with TRC105, an endoglin-targeting antibody. In this study, we investigated the combination of endoglin antibodies (TRC105 and M1043) with an anti-PD1 antibody.Experimental Design:The combination anti-endoglin/anti-PD1 antibodies was tested in four preclinical mouse models representing different stages of cancer development. To investigate the underlying mechanism, Fc-receptor–knockout mice were used complemented with depletion of multiple immune subsets in mice. Tumor growth and the composition of immune infiltrate were analyzed by flow cytometry. Finally, human colorectal cancer specimens were analyzed for presence of endoglin-expressing regulatory T cells (Treg).Results:In all models, the combination of endoglin antibody and PD1 inhibition produced durable tumor responses, leading to complete regressions in 30% to 40% of the mice. These effects were dependent on the presence of Fcγ receptors, indicating the involvement of antibody-dependent cytotoxic responses and the presence of CD8+ cytotoxic T cells and CD4+ Th cells. Interestingly, treatment with the endoglin antibody, TRC105, significantly decreased the number of intratumoral Tregs. Endoglin-expressing Tregs were also detected in human colorectal cancer specimens.Conclusions:Taken together, these data provide a rationale for combining TRC105 and anti-PD1 therapy and provide additional evidence of endoglin's immunomodulatory role.

The medians and ranges for all analytes at baseline.

Purpose:GOG-0218, a double-blind placebo-controlled phase III trial, compared carboplatin and paclitaxel with placebo, bevacizumab followed by placebo, or bevacizumab followed by bevacizumab in advanced epithelial ovarian cancer (EOC). Results demonstrated significantly improved progression-free survival (PFS), but no overall survival (OS) benefit with bevacizumab. Blood samples were collected for biomarker analyses.Experimental Design:Plasma samples were analyzed via multiplex ELISA technology for seven prespecified biomarkers [IL6, Ang-2, osteopontin (OPN), stromal cell–derived factor-1 (SDF-1), VEGF-D, IL6 receptor (IL6R), and GP130]. The predictive value of each biomarker with respect to PFS and OS was assessed using a protein marker by treatment interaction term within the framework of a Cox proportional hazards model. Prognostic markers were identified using Cox models adjusted for baseline covariates.Results:Baseline samples were available from 751 patients. According to our prespecified analysis plan, IL6 was predictive of a therapeutic advantage with bevacizumab for PFS (P = 0.007) and OS (P = 0.003). IL6 and OPN were found to be negative prognostic markers for both PFS and OS (P < 0.001). Patients with high median IL6 levels (dichotomized at the median) treated with bevacizumab had longer PFS (14.2 vs. 8.7 months) and OS (39.6 vs. 33.1 months) compared with placebo.Conclusions:The inflammatory cytokine IL6 may be predictive of therapeutic benefit from bevacizumab when combined with carboplatin and paclitaxel. Aligning with results observed in patients with renal cancer treated with antiangiogenic therapies, it appears plasma IL6 may also define those patients with EOC more or less likely to benefit from the addition of bevacizumab to standard chemotherapy.

Supplemental Figure 1. Changes in average vascular score (AVS) from baseline.

Supplementary Figure 4A. Kaplan-Meier PFS curves by treatment and dichotomized angiokines groups for following angiokines: TGFb-R3, HGF, TSP-2, OPN, IL-6, SDF-1, Ang-2, ICAM-1, TGF-b2, TIMP-1, BMP-9, PDGF-BB, VCAM-1, PDGF-AA, VEGF-D, TGF-b1, VEGF-R1, HER-3, VEGF, PlGF, CD-73, VEGF-R3, and VEGF-R2

Supplementary Table from Plasma Protein Biomarkers in Advanced or Metastatic Colorectal Cancer Patients Receiving Chemotherapy With Bevacizumab or Cetuximab: Results from CALGB 80405 (Alliance)

Supplemental Table 3. BEP study patients and the number of patients whose granulation tissue biopsies were available for biomarker study.

Purpose:Preclinical studies suggest PARP inhibition (PARPi) induces immunostimulatory micromilieu in ovarian cancer thus complementing activity of immune checkpoint blockade. We conducted a phase II trial of PARPi olaparib and anti–PD-L1 durvalumab and collected paired fresh core biopsies and blood samples to test this hypothesis.Patients and Methods:In a single-center, proof-of-concept phase II study, we enrolled women aged ≥18 with recurrent ovarian cancer. All patients were immune checkpoint inhibitor–naïve and had measurable disease per RECISTv1.1, ECOG performance status 0–2, and adequate organ and marrow function. Patients received olaparib 300 mg twice daily and durvalumab 1,500 mg intravenously every 4 weeks until disease progression, unacceptable toxicity, or withdrawal of consent. Primary endpoint was overall response rate (ORR). Secondary objectives were safety and progression-free survival (PFS). Translational objectives included biomarker evaluation for relationships with clinical response and immunomodulatory effects by treatment.Results:Thirty-five patients with ovarian cancer [median, four prior therapies (IQR, 2–5.5), predominantly platinum-resistant (86%), BRCA wild-type (77%)] received at least one full cycle of treatment. ORR was 14% [5/35; 95% confidence interval (CI), 4.8%–30.3%]. Disease control rate (PR+SD) was 71% (25/35; 95% CI, 53.7%–85.4%). Treatment enhanced IFNγ and CXCL9/CXCL10 expression, systemic IFNγ/TNFα production, and tumor-infiltrating lymphocytes, indicating an immunostimulatory environment. Increased IFNγ production was associated with improved PFS [HR, 0.37 (95% CI, 0.16–0.87), P = 0.023], while elevated VEGFR3 levels were associated with worse PFS (HR, 3.22 (95% CI, 1.23–8.40), P = 0.017].Conclusions:The PARPi and anti–PD-L1 combination showed modest clinical activity in recurrent ovarian cancer. Our correlative study results suggest immunomodulatory effects by olaparib/durvalumab in patients and indicate that VEGF/VEGFR pathway blockade would be necessary for improved efficacy of the combination.

Supplementary Figure 2. Kaplan-Meier PFS curves by dichotomized angiokines: CD-73, BMP-9, VEGF-R1, PDGF-BB, VEGF, IL-6, VCAM-1, OPN, HGF, PDGF-AA, TGFb-R3, TGF-b1, VEGF-R3, and TGF-b2

Relationship between covariates and IL6.

CONSORT diagram

Purpose:CALGB 90206 was a phase III trial of 732 patients with metastatic renal cell carcinoma (mRCC) comparing bevacizumab plus IFNα (BEV + IFN) with IFNα alone (IFN). No difference in overall survival (OS) was observed. Baseline samples were analyzed to identify predictive biomarkers for survival benefit.Patients and Methods:A total of 32 biomarkers were assessed in 498 consenting patients randomly assigned into training (n = 279) and testing (n = 219) sets. The proportional hazards model was used to test for treatment arm and biomarker interactions of OS. The estimated coefficients from the training set were used to compute a risk score for each patient and to classify patients by risk in the testing set. The resulting model was assessed for predictive accuracy using the time-dependent area under the ROC curve (tAUROC).Results:A statistically significant three-way interaction between IL6, hepatocyte growth factor (HGF), and bevacizumab treatment was observed in the training set and confirmed in the testing set (P < 0.0001). The model based on IL6, HGF, and bevacizumab treatment was predictive of OS (P < 0.001), with the high- and low-risk groups having a median OS of 10.2 [95% confidence interval (CI), 8.0–13.8] and 34.3 (95% CI, 28.5–40.5) months, respectively. The average tAUROC for the final model of OS based on 100 randomly split testing sets was 0.78 (first, third quartiles = 0.77, 0.79).Conclusions:IL6 and HGF are potential predictive biomarkers of OS benefit from BEV + IFN in patients with mRCC. The model based on key biological and clinical factors demonstrated predictive efficacy for OS. These markers warrant further validation in future anti-VEGF and immunotherapy in mRCC trials.See related commentaries by Mishkin and Kohn, p. 2722 and George and Bertagnolli, p. 2725

Legends

Purpose: In early clinical testing, most novel targeted anticancer therapies have limited toxicities and limited efficacy, which complicates dose and schedule selection for these agents. Confirmation of target inhibition is critical for rational drug development; however, repeated tumor biopsies are often impractical and peripheral blood mononuclear cells and normal skin are often inadequate surrogates for tumor tissue. Based upon the similarities of tumor and wound stroma, we have developed a clinical dermal granulation tissue model to evaluate novel targeted therapies.Experimental Design: A 4-mm skin punch biopsy was used to stimulate wound healing and a repeat 5-mm punch biopsy was used to harvest the resulting granulation tissue. This assay was performed at pretreatment and on-treatment evaluating four targeted therapies, bevacizumab, everolimus, erlotinib, and panitumumab, in the context of three different clinical trials. Total and phosphorylated levels VEGFR2, S6RP, and EGFR were evaluated using ELISA-based methodologies.Results: Significant and consistent inhibition of the VEGF pathway (using VEGFR2 as the readout) was observed in granulation tissue biopsies from patients treated with bevacizumab and everolimus. In addition, significant and consistent inhibition of the mTOR pathway (using S6RP as the readout) was observed in patients treated with everolimus. Finally, significant inhibition of the EGFR pathway (using EGFR as the readout) was observed in patients treated with panitumumab, but this was not observed in patients treated with erlotinib.Conclusions: Molecular analyses of dermal granulation tissue can be used as a convenient and quantitative pharmacodynamic biomarker platform for multiple classes of targeted therapies. Clin Cancer Res; 21(15); 3442–52. ©2015 AACR.

Supplementary Figure 3. Kaplan-Meier OS curves by dichotomized angiokines: VEGF-D, VEGF, VEGF-R1, PDGF-AA, TGF-b1, VEGF-R3, HER-3, BMP-9, CD-73, VEGF-R2, TGF-b2, SDF-1, PDGF-BB, and TGFb-R3

Purpose: Formalin-fixed, paraffin-embedded tumor samples from CALGB 80203 were analyzed for expression of EGFR axis–related genes to identify prognostic or predictive biomarkers for cetuximab treatment.Patients and Methods: Patients (238 total) with first-line metastatic colorectal cancer (mCRC) were randomized to FOLFOX or FOLFIRI chemotherapy ± cetuximab. qRT-PCR analyses were conducted on tissues from 103 patients at baseline to measure gene expression levels of HER-related genes, including amphiregulin (AREG), betacellulin (BTC), NT5E (CD73), DUSP4, EGF, EGFR, epigen (EPGN), epiregulin (EREG), HBEGF, ERBB2 (HER2), ERBB3 (HER3), ERBB4 (HER4), PHLDA1, and TGFA. The interactions between expression levels and treatment with respect to progression-free survival (PFS) and overall survival (OS) were modeled using multiplicative Cox proportional hazards models.Results: High tumor mRNA levels of HER2 [hazard ratio (HR), 0.64; P = 0.002] and EREG (HR, 0.89; P = 0.016) were prognostic markers associated with longer PFS across all patients. HER3 and CD73 expression levels were identified as potential predictive markers of benefit from cetuximab. In KRAS wild-type (WT) tumors, low HER3 expression was associated with longer OS from cetuximab treatment, whereas high HER3 expression was associated with shorter OS from cetuximab treatment (chemo + cetuximab: HR, 1.15; chemo-only: HR, 0.48; Pinteraction = 0.029). High CD73 expression was associated with longer PFS from cetuximab treatment in patients with KRAS-WT (chemo + cetuximab: HR, 0.91; chemo-only: HR, 1.57; Pinteraction = 0.026) and KRAS-mutant (Mut) tumors (chemo + cetuximab: HR, 0.80; chemo-only: HR, 1.29; P = 0.025).Conclusions: Gene expression of HER3 and CD73 was identified as a potential predictive marker for cetuximab. These data implicate HER axis signaling and immune modulation as potential mechanisms of cetuximab action and sensitivity. Clin Cancer Res; 21(5); 1078–86. ©2014 AACR.

3-way interaction analyses between outcome, treatment, and markers.

Supplemental Table 1. Characteristics of patients whose granulation tissue biopsies were available for biomarker analysis and patients whose average vascular score (AVS) were available.

Purpose:CALGB 80405 compared the combination of first-line chemotherapy with cetuximab or bevacizumab in the treatment of advanced or metastatic colorectal cancer (mCRC). Although similar clinical outcomes were observed in the cetuximab-chemotherapy group and the bevacizumab-chemotherapy group, biomarkers could identify patients deriving more benefit from either biologic agent.Patients and Methods:In this exploratory analysis, the Angiome, a panel of 24 soluble protein biomarkers were measured in baseline plasma samples in CALGB 80405. Prognostic biomarkers were determined using univariate Cox proportional hazards models. Predictive biomarkers were identified using multivariable Cox regression models including interaction between biomarker level and treatment.Results:In the total population, high plasma levels of Ang-2, CD73, HGF, ICAM-1, IL6, OPN, TIMP-1, TSP-2, VCAM-1, and VEGF-R3 were identified as prognostic of worse progression-free survival (PFS) and overall survival (OS). PlGF was identified as predictive of lack of PFS benefit from bevacizumab [bevacizumab HR, 1.51; 95% confidence interval (CI), 1.10–2.06; cetuximab HR, 0.94; 95% CI, 0.71–1.25; Pinteraction = 0.0298] in the combined FOLFIRI/FOLFOX regimens. High levels of VEGF-D were predictive of lack of PFS benefit from bevacizumab in patients receiving FOLFOX regimen only (FOLFOX/bevacizumab HR, 1.70; 95% CI, 1.19–2.42; FOLFOX/cetuximab HR, 0.92; 95% CI, 0.68–1.24; Pinteraction = 0.0097).Conclusions:In this exploratory, hypothesis-generating analysis, the Angiome identified multiple prognostic biomarkers and two potential predictive biomarkers for patients with mCRC enrolled in CALGB 80405. PlGF and VEGF-D predicted lack of benefit from bevacizumab in a chemo-dependent manner.See related commentaries by Mishkin and Kohn, p. 2722 and George and Bertagnolli, p. 2725

Supplementary figures S1-S5