Supplementary Figure S1 shows biomarker variability over time.

Supplementary Figure S2 show intra- and inter-participants variability.

Abstract Background: Biomarker analyses are an integral part of cancer research. Despite the intense efforts to identify and characterize biomarkers in patients with cancer, little is known regarding the natural variation of biomarkers in healthy populations. Here we conducted a clinical study to evaluate the natural variability of biomarkers over time in healthy participants. Methods: The angiome multiplex array, a panel of 25 circulating protein biomarkers, was assessed in 28 healthy participants across eight timepoints over the span of 60 days. We utilized the intraclass correlation coefficient (ICC) to quantify the reliability of the biomarkers. Adjusted ICC values were calculated under the framework of a linear mixed-effects model, taking into consideration age, sex, body mass index, fasting status, and sampling factors. Results: ICC was calculated to determine the reliability of each biomarker. Hepatocyte growth factor was the most stable marker (ICC = 0.973), while platelet-derived growth factor (PDGF)-BB was the most variable marker (ICC = 0.167). In total, ICC analyses revealed that 22 out of 25 measured biomarkers display good (≥0.4) to excellent (>0.75) ICC values. Three markers (PDGF-BB, TGFβ1, PDGF-AA) had ICC values <0.4. Greater age was associated with higher IL6 (P = 0.0114). Higher body mass index was associated with higher levels of IL6 (P = 0.0003) and VEGF-R3 (P = 0.0045). Conclusions: Of the 25 protein biomarkers measured over this short time period, 22 markers were found to have good or excellent ICC values, providing additional validation for this biomarker assay. Impact: These data further support the validation of the angiome biomarker assay and its application as an integrated biomarker in clinical trial testing.

Supplementary Figure S3 shows homoscedasticity and normality of residuals.

Objective:This pilot window of opportunity study was conducted to assess feasibility, toxicity, and changes in immune parameters in response to one dose of the PD-1 inhibitor, pembrolizumab, in patients newly diagnosed with mullerian epithelial cancers. Methods:Eligible patients received pembrolizumab 200 mg IV once ≥ 7 days prior to further standard therapy, including adjuvant chemotherapy. Tissue and blood were collected before and ≥ 7 days after pembrolizumab administration. Primary endpoints included change in tumor infiltrating lymphocytes (TIL), feasibility, and toxicity based on frequency and severity of adverse events. Exploratory objectives included tumor assessment of immunohistochemical PD-L1 staining using a quantitative modified proportion score and qualitative assessment of immune presence at the stromal interface. Measurement of cytokine levels and digital spatial profiling were performed from plasma and tissue samples, respectively, before and after pembrolizumab. Results:Fifteen patients enrolled and received pembrolizumab. TIL levels changed in 4 of 11 paired sets, with 3 decreasing and 1 increasing post-treatment. PD-L1 modified proportion score increased in 7 cases, decreased in 2, and remained unchanged in 2. The stromal interface switched from negative to positive in 3 cases. Collectively, 8 of 10 assessable tumor pairs demonstrated either an increase in PD-L1 modified proportion score or the stromal interface switched from negative to positive. Circulating CXCL10 and TNFα levels increased after pembrolizumab in patients with response, but decreased in the one patient with progression on adjuvant chemotherapy. Digital spatial profiling showed increased IDO1 protein expression in immune and tumor compartments after treatment. Conclusion:A single dose of pembrolizumab increased PD-L1 modified proportion score and/or stromal interface immune cells suggesting potential for local tumor immunologic recruitment. Additionally, increases in systemic inflammation, measured by cytokine production and differential IDO1 expression, reflect an interferon response. These hypothesis-generating data need to be confirmed and validated in larger subsets.

Since multiple front-line immune checkpoint inhibitor-based (ICI-based) combinations are approved for metastatic renal cell carcinoma, biomarkers predicting for ICI responses are needed past clinical prognostication scores and transcriptome gene expression profiling. Circulating markers represent opportunities to assess baseline and dynamic changes in immune cell frequency and cytokine levels while on treatment. We conducted an exploratory prospective correlative study of 33 patients with metastatic clear cell renal cell carcinoma undergoing treatment with ICIs and correlated changes in circulating immune cell subsets and cytokines with clinical responses to treatment. Cell frequencies and cytokine levels were compared between responders and nonresponders using unpaired parametric t tests, using prespecified alpha level of significance of 0.05. Classical monocyte subsets (CD14+CD16-), as well as 7 cytokines (IL-12/23 p40, macrophage inflammatory protein-1a, macrophage inflammatory protein-1b, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, IL-8, and TNF-α) were higher at baseline for responding versus nonresponding patients. Dynamic changes in thymus- and activation-regulated chemokine (TARC), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) also correlated with patients with ICI response. In summary, macrophage-activating agents were observed to be important in ICI response and may highlight the importance of the innate immune response in ICI responses.

In pathologies including cancer, aberrant Transforming Growth Factor-β (TGF-β) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 1–3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-β signaling and the cells' responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-β signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs’, which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.

Background CALGB 90401 (Alliance) was a phase III trial of 1050 patients with metastatic castration-resistant prostate cancer (mCRPC) comparing docetaxel, prednisone, bevacizumab (DP+B) versus DP alone. While this trial did not show an improvement in overall survival (OS), there were improved intermediate outcomes suggesting that subsets of men may derive benefit from this combination. The purpose of this analysis was to identify prognostic and predictive biomarkers associated with OS and progression-free survival (PFS) benefit from DP+B. Methods Baseline EDTA plasma samples from 650 consenting patients were analyzed for 24 biomarkers. The proportional hazards model was utilized to test for the prognostic and predictive importance of the biomarkers for OS. The statistically significant biomarkers of OS were further investigated for prognostic and predictive importance for other secondary outcomes. Results 15 markers [ICAM-1, VEGF-R3, TIMP-1, TSP-2, Ang-2, Her-3, Osteopontin (OPN), PlGF, VCAM-1, HGF, VEGF, Chromogranin A, IL-6, VEGF-R1, BMP-9] were prognostic of OS, while 9 markers (ICAM-1, VEGF-R3, Her-3, TIMP-1, Ang-2, OPN, PlGF, HGF, and VEGF) were also prognostic of PFS. All markers were statistically significant in univariate analyses after adjustment for multiplicity (FDR < 0.1). In multivariable analyses of OS adjusting for risk score, seven markers had FDR < 0.1, including ICAM-1, VEGF-R3, TIMP-1, Ang-2, VEGF, TSP-2 and HGF. In unadjusted analysis, OPN was predictive of PFS improvement with DP+B, in both univariate and multivariable analysis. However, none of the biomarkers tested were predictive of clinical outcomes after adjusting for multiple comparisons. Conclusions Multiple biomarkers were identified in CALGB 90401 as prognostic of clinical outcomes but not predictive of OS. While OPN may have promise as a potential biomarker for anti-angiogenic therapies, further mechanistic and clinical studies are needed to determine the underlying biology and potential clinical application.

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

Quartile analysis of IL6. Median survival times are given in months, and HR's represent treatment hazard ratios.

Biomarkers change on treatment

Flow diagram of patients through the study.

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.

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).

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.

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

Relationship between covariates and IL6.

Association between outcomes (PFS and OS) and baseline covariates (age, stage/debulking status, and performance status).

In pathologies such as cancer, aberrant Transforming Growth Factor-β (TGF-β) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pivotal pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 1-3 and inhibin A/B. While betaglycan can be membrane-bound, it can also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. The extracellular domain of betaglycan undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. Here we report the unexpected discovery that the heparan sulfate modifications are critical for the ectodomain shedding of betaglycan. In the absence of such modifications, betaglycan is not shed. Such shedding is indispensable for the ability of betaglycan to suppress TGF-β signaling and the cells' responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key regulator of betaglycan shedding and thereby TGF-β signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan modifications of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs', which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.

The medians and ranges for all analytes at baseline.