Common variation in a long non-coding RNA gene modulates variation of circulating TGF-ß2 levels in metastatic colorectal cancer patients (Alliance).

BACKGROUND: Herein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC). The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings. RESULTS: The final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-ß2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73. CONCLUSIONS: This study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-ß2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.

Prognostic and predictive analyses of circulating plasma biomarkers in men with metastatic castration resistant prostate cancer treated with docetaxel/prednisone with or without bevacizumab.

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.

Common variation in a long non-coding RNA gene modulates variation of circulating TGF-ß2 levels in metastatic colorectal cancer patients (Alliance).

Background: Herein, we report results from a genome-wide study conducted to identify protein quantitative trait loci (pQTL) for circulating angiogenic and inflammatory protein markers in patients with metastatic colorectal cancer (mCRC).The study was conducted using genotype, protein marker, and baseline clinical and demographic data from CALGB/SWOG 80405 (Alliance), a randomized phase III study designed to assess outcomes of adding VEGF or EGFR inhibitors to systemic chemotherapy in mCRC patients. Germline DNA derived from blood was genotyped on whole-genome array platforms. The abundance of protein markers was quantified using a multiplex enzyme-linked immunosorbent assay from plasma derived from peripheral venous blood collected at baseline. A robust rank-based method was used to assess the statistical significance of each variant and protein pair against a strict genome-wide level. A given pQTL was tested for validation in two external datasets of prostate (CALGB 90401) and pancreatic cancer (CALGB 80303) patients. Bioinformatics analyses were conducted to further establish biological bases for these findings. Results: The final analysis was carried out based on data from 540,021 common typed genetic variants and 23 protein markers from 869 genetically estimated European patients with mCRC. Correcting for multiple testing, the analysis discovered a novel cis-pQTL in LINC02869, a long non-coding RNA gene, for circulating TGF-ß2 levels (rs11118119; AAF = 0.11; P-value < 1.4e-14). This finding was validated in a cohort of 538 prostate cancer patients from CALGB 90401 (AAF = 0.10, P-value < 3.3e-25). The analysis also validated a cis-pQTL we had previously reported for VEGF-A in advanced pancreatic cancer, and additionally identified trans-pQTLs for VEGF-R3, and cis-pQTLs for CD73. Conclusions: This study has provided evidence of a novel cis germline genetic variant that regulates circulating TGF-ß2 levels in plasma of patients with advanced mCRC and prostate cancer. Moreover, the validation of previously identified pQTLs for VEGF-A, CD73, and VEGF-R3, potentiates the validity of these associations.

Hypoxia-induced inhibin promotes tumor growth and vascular permeability in ovarian cancers.

Hypoxia, a driver of tumor growth and metastasis, regulates angiogenic pathways that are targets for vessel normalization and ovarian cancer management. However, toxicities and resistance to anti-angiogenics can limit their use making identification of new targets vital. Inhibin, a heteromeric TGFß ligand, is a contextual regulator of tumor progression acting as an early tumor suppressor, yet also an established biomarker for ovarian cancers. Here, we find that hypoxia increases inhibin levels in ovarian cancer cell lines, xenograft tumors, and patients. Inhibin is regulated primarily through HIF-1, shifting the balance under hypoxia from activins to inhibins. Hypoxia regulated inhibin promotes tumor growth, endothelial cell invasion and permeability. Targeting inhibin in vivo through knockdown and anti-inhibin strategies robustly reduces permeability in vivo and alters the balance of pro and anti-angiogenic mechanisms resulting in vascular normalization. Mechanistically, inhibin regulates permeability by increasing VE-cadherin internalization via ACVRL1 and CD105, a receptor complex that we find to be stabilized directly by inhibin. Our findings demonstrate direct roles for inhibins in vascular normalization via TGF-ß receptors providing new insights into the therapeutic significance of inhibins as a strategy to normalize the tumor vasculature in ovarian cancer.

Plasma levels of VEGF-A and VCAM-1 as predictors of drug-induced hypertension in patients treated with VEGF pathway inhibitors.

AIMS: Hypertension is a common toxicity induced by vascular endothelial growth factor (VEGF) pathway inhibitors. There are no validated markers of hypertension induced by these drugs. METHODS: We previously discovered that cancer patients with lower plasma levels of angiopoietin-2, VCAM-1 and VEGF-A are at high risk of developing severe hypertension when treated with bevacizumab. This study aimed to validate the predictive value of these markers in pretreatment plasma samples of an additional cohort of 101 colorectal cancer patients treated with regorafenib. The levels of angiopoietin-2, VCAM-1 and VEGF-A were measured by enzyme-linked immunosorbent assay (ELISA). The association between proteins and grade ≥2 regorafenib-induced hypertension was performed by calculating the odds ratio (OR) from logistic regression. Using the optimal cut-point of each protein, sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for hypertension were estimated. RESULTS: Lower levels of VCAM-1 (P = .015, OR = 3.11, 95% CI 1.27-8.08) and VEGF-A (P = .007, OR = 3.47, 95% CI 1.40-8.75) were associated with a higher risk of hypertension. Levels of angiopoietin-2 were not associated with hypertension. The multivariable model indicates an independent effect of VCAM-1 (P = .018, OR = 3.18, 95% CI 1.25-8.68) and VEGF-A (P = .008, OR = 3.77, 95% CI 1.44-10.21). The presence of low levels of both VCAM-1 and VEGF-A had an OR of 9.46 (95% CI 3.08-33.26, P = 1.70 × 10-4 ) for the risk of hypertension (sensitivity of 41.4%, specificity of 93.1%, PPV of 70.6% and NPV of 79.8%). CONCLUSIONS: This study confirmed the value of VCAM-1 and VEGF-A levels in predicting hypertension induced by regorafenib, another VEGF pathway inhibitor.

Phase I Study of Lenvatinib and Capecitabine with External Radiation Therapy in Locally Advanced Rectal Adenocarcinoma.

BACKGROUND: Neoadjuvant chemoradiation with fluoropyrimidine followed by surgery and adjuvant chemotherapy has been the standard treatment of locally advanced stages II and III rectal cancer for many years. There is a high risk for disease recurrence; therefore, optimizing chemoradiation strategies remains an unmet need. Based on a few studies, there is evidence of the synergistic effect of VEGF/PDGFR blockade with radiation. METHODS: In this phase I, dose-escalation and dose-expansion study, we studied 3 different dose levels of lenvatinib in combination with capecitabine-based chemoradiation for locally advanced rectal cancer. RESULTS: A total of 20 patients were enrolled, and 19 were eligible for assessment of efficacy. The combination was well tolerated, with an MTD of 24 mg lenvatinib. The downstaging rate for the cohort and the pCR was 84.2% and 37.8%, respectively. Blood-based protein biomarkers TSP-2, VEGF-R3, and VEGF correlated with NAR score and were also differentially expressed between response categories. The NAR, or neoadjuvant rectal score, encompasses cT clinical tumor stage, pT pathological tumor stage, and pN pathological nodal stage and provides a continuous variable for evaluating clinical trial outcomes. CONCLUSION: The combination of lenvatinib with capecitabine and radiation in locally advanced rectal cancer was found to be safe and tolerable, and potential blood-based biomarkers were identified. CLINICAL TRIAL REGISTRATION: NCT02935309.

Cabozantinib with or without Panitumumab for RAS wild-type metastatic colorectal cancer: impact of MET amplification on clinical outcomes and circulating biomarkers.

PURPOSE: Acquired resistance to EGFR inhibitors in metastatic colorectal cancer (mCRC) remains a hurdle for effective treatment. MET amplification has been indicated as a driver of acquired resistance. Clinical activity has been demonstrated for the combination of EGFR and MET inhibitors in mCRC. But the impact of this regimen on angiogenesis and inflammation remains largely unknown. METHODS: In this non-randomized, open-label phase Ib/II study, four patients were treated with cabozantinib alone and 25 patients received the combination of cabozantinib and panitumumab. MET amplification was detected in blood in all four patients treated with cabozantinib monotherapy and 5/25 patients treated with cabozantinib and panitumumab combination therapy. Plasma samples from 28 patients were available for biomarker analysis. RESULTS: A panel of circulating protein biomarkers was assessed in patient plasma at baseline and on-treatment. Baseline marker levels were analyzed for prognostic value for clinical outcomes, including MET amplification as a covariate. HGF and OPN were prognostic for both progression-free survival (PFS) and overall survival (OS), while six markers (IL-6, VCAM-1, VEGF-R1, TSP-2, TIMP-1, ICAM-1) were prognostic only for OS. In patients with MET amplification, baseline PDGF-AA, PDGF-BB, TGF-ß1, and VEGF-C levels were significantly higher, whereas baseline TGFß-R3 levels were significantly lower than MET non-amplified patients. On-treatment change of four markers (CD73, PlGF, PDGF-BB, VEGF) were significantly different between MET amplified and non-amplified subpopulations. CONCLUSION: This study identified circulating HGF and several inflammatory and angiogenic proteins as prognostic biomarkers. Furthermore, MET amplification status is associated with both baseline expression and on-treatment modulation of members of angiogenesis and TGF-ß pathway proteins. CLINICAL TRIALS REGISTRATION NUMBER: ClinicalTrials.gov identifier: NCT02008383.

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

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.

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

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.

Microvesicles, blood cells, and endothelial cells mediate phosphatidylserine-related prothrombotic state in patients with periodontitis.

BACKGROUND: Phosphatidylserine (PS) is essential for inflammation-associated thrombogenesis, but the exact effect of PS on the prothrombotic state in periodontitis is uncertain. This study aimed to determine the PS-related procoagulant state in patients with periodontitis. METHODS: A total of 138 patients with periodontitis were examined compared with 42 healthy controls. PS-exposing cells and microvesicles in blood samples were detected by confocal microscopy and flow cytometry. The clotting time assay and prothrombinase complex formation assay were used to measure the procoagulant activity of microvesicles, blood cells and endothelial cells. Periodontal clinical parameters and laboratory characteristics of patients with severe periodontitis were recorded and analyzed at baseline and 6 months after non-surgical periodontal therapy. RESULTS: Total PS-positive (PS+ ) microvesicles and the percentage of PS+ blood cells increased in patients with severe periodontitis compared with patients with moderate/mild periodontitis or healthy controls. Endothelial cells cultured in serum from patients with severe periodontitis expressed more PS compared with those cultured in serum from healthy controls. Specifically, PS exposure on blood cells and endothelial cells significantly decreased after inhibiting the effect of inflammatory cytokines. The elevated levels of PS+ cells and microvesicles in severe periodontitis shortened clotting time and led to increased prothrombinase complex formation. Non-surgical periodontal therapy significantly attenuated the release of microvesicles and the PS exposure of blood cells in severe periodontitis. CONCLUSIONS: The prothrombotic state of patients with periodontitis is mediated by PS+ cells and microvesicles stimulated by elevated levels of inflammatory cytokines.

Plasma levels of angiopoietin-2, VEGF-A, and VCAM-1 as markers of bevacizumab-induced hypertension: CALGB 80303 and 90401 (Alliance).

Hypertension is a common toxicity induced by bevacizumab and other antiangiogenic drugs. There are no biomarkers to predict the risk of bevacizumab-induced hypertension. This study aimed to identify plasma proteins related to the function of the vasculature to predict the risk of severe bevacizumab-induced hypertension. Using pretreated plasma samples from 398 bevacizumab-treated patients in two clinical trials (CALGB 80303 and 90401), the levels of 17 proteins were measured via ELISA. The association between proteins and grade 3 bevacizumab-induced hypertension was performed by calculating the odds ratio (OR) from logistic regression adjusting for age, sex, and clinical trial. Using the optimal cut-point of each protein, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for hypertension were estimated. Five proteins showed no difference in levels between clinical trials and were used for analyses. Lower levels of angiopoietin-2 (p = 0.0013, OR 3.41, 95% CI 1.67-7.55), VEGF-A (p = 0.0008, OR 4.25, 95% CI 1.93-10.72), and VCAM-1 (p = 0.0067, OR 2.68, 95% CI 1.34-5.63) were associated with an increased risk of grade 3 hypertension. The multivariable model suggests independent effects of angiopoietin-2 (p = 0.0111, OR 2.71, 95% CI 1.29-6.10), VEGF-A (p = 0.0051, OR 3.66, 95% CI 1.54-9.73), and VCAM-1 (p = 0.0308, OR 2.27, 95% CI 1.10-4.92). The presence of low levels of 2-3 proteins had an OR of 10.06 (95% CI 3.92-34.18, p = 1.80 × 10-5) for the risk of hypertension, with sensitivity of 89.7%, specificity of 53.5%, PPV of 17.3%, and NPV of 97.9%. This is the first study providing evidence of plasma proteins with potential value to predict patients at risk of developing bevacizumab-induced hypertension.Clinical trial registration: ClinicalTrials.gov Identifier: NCT00088894 (CALGB 80303); and NCT00110214 (CALGB 90401).

Design and optimizing a new CDP-choline in vitro multienzyme producing process starts from d-ribose.

This work designs an in vitro multienzyme system to produce CDP-choline from d-ribose and develop an optimization procedure for one-pot multienzyme catalytic system. The entire process integrated 10 enzymes, and an efficient acetate kinase/acetyl phosphate-based ATP regeneration module was applied. Then, some optimizations to this system were made including selecting optimum enzyme building blocks and improving expression parameters. The process improved the final yield of CDP-choline from 0.2 to 6 g/L (CDP-choline titer 12.2 mM). This new one-pot CDP-choline producing system has a potential for industrial use, and the optimization procedure shed light on improving other one-pot multienzyme system for industrial production of energy rich compounds.

Pilot investigation of circulating angiogenic and inflammatory biomarkers associated with vascular malformations.

BACKGROUND: Vascular malformations in the central nervous system are difficult to monitor and treat due to their inaccessible location. Angiogenic and inflammatory proteins are secreted into the bloodstream and may serve as useful biomarkers for identifying patients at risk for complications or with certain disease phenotypes. METHODS: A validated multiplex protein array consisting of 26 angiogenic and inflammatory biomarkers (Angiome) was assessed in plasma isolated from healthy controls and patients with either sporadic brain arteriovenous malformation (BAVM), familial cerebral cavernous malformation (CCM), or hereditary hemorrhagic telangiectasia (HHT). These samples were obtained from archives of ongoing research studies at the University of California San Francisco and through prospective collection at the Toronto HHT Centre at St. Michael's Hospital. RESULTS: We compared circulating biomarker levels from each patient group to healthy controls and analyzed each pairwise combination of patient groups for differences in biomarker levels. Additionally, we analyzed the HHT samples to determine the association between biomarker levels and the following HHT-specific phenotypes, BAVM, pulmonary arteriovenous malformation (PAVM), liver vascular malformation (LVM), and gastrointestinal (GI) bleeding. Compared to controls, levels of SDF1 were significantly elevated in HHT patients (Proportional Increase [PI] = 1.87, p < 0.001, q = 0.011). Levels of sENG were significantly reduced in HHT patients compared to controls (PI = 0.56, p < 0.001, q < 0.001), reflecting the prevalence of HHT1 patients in this cohort. Levels of IL6 (PI = 3.22, p < 0.001, q < 0.001) and sTGFßR3 (PI = 0.70, p = 0.001, q < 0.029) differed significantly in CCM patients compared to controls. Compared to controls, ten of the biomarkers were significantly different in sporadic BAVM patients (q-values < 0.05). Among the pairwise combinations of patient groups, a significant elevation was observed in TGFß1 in CCM patients compared to sporadic BAVM patients (PI = 2.30, p < 0.001, q = 0.034). When examining the association of circulating biomarker levels with HHT-specific phenotypes, four markers were significantly lower in HHT patients with BAVM (q-values < 0.05), and four markers were significantly higher in patients with LVM (q-values < 0.05). CONCLUSIONS: This pilot study suggests that the profile of circulating angiogenic and inflammatory biomarkers may be unique to each type of vascular malformation. Furthermore, this study indicates that circulating biomarkers may be useful for assessing phenotypic traits of vascular malformations.

A Procedure to Design One-Pot Multi-enzyme System for Industrial CDP-Choline Production.

Fermentation and chemical methods for industrial cytidine diphosphate choline (CDP-choline) catalytic production both suffer from several disadvantages such as relatively low efficiency and productivity. To overcome these problems, we applied the concept of synthetic biology to develop a new one-pot multi-enzyme system to produce CDP-choline from orotic acid. Enzymes from different sources were selected and optimized as building blocks of the system, and parameters such as oxygen supply were also optimized. This system shows a titer of 37.6 ± 1.1 mM and a reaction rate of 1.6 mM L-1 h-1, both increase 66 % from traditional processes. It also has an efficiency of energy of 25.4%, improves 2-folds. This new one-pot CDP-choline-producing system has a potential for industrial use, and the procedure to design one-pot multi-enzyme system can be applied to build other one-pot system producing energy-rich compounds.

Phosphatidylserine-exposing tumor-derived microparticles exacerbate coagulation and cancer cell transendothelial migration in triple-negative breast cancer.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations. Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay. Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells. Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Clinical Results and Biomarker Analyses of Axitinib and TRC105 versus Axitinib Alone in Patients with Advanced or Metastatic Renal Cell Carcinoma (TRAXAR).

LESSONS LEARNED: The combination of carotuximab with axitinib did not provide a benefit over axitinib monotherapy in patients with metastatic clear cell renal cell carcinoma who had previously progressed on one or more vascular endothelial growth factor (VEGF)-targeted therapies. Exploratory evaluation of pretreatment circulating biomarkers suggested the combination might benefit patients who have low baseline VEGF levels. BACKGROUND: Endoglin is an angiogenic receptor expressed on proliferating tumor vessels and renal cell carcinoma (RCC) stem cells that is implicated as a mechanism of resistance to vascular endothelial growth factor receptor (VEGFR) inhibitors. This study evaluated an antiendoglin monoclonal antibody (carotuximab, TRC105) combined with axitinib in patients with advanced or metastatic clear cell renal cell carcinoma (mccRCC) who had progressed following one or more prior VEGF inhibitors. METHODS: TRAXAR was a multicenter, international randomized 1:1 (stratified by ECOG, 0 vs. 1), phase II study of carotuximab combined with axitinib versus axitinib alone in mccRCC patients who had progressed following one or more prior VEGF inhibitors. The primary endpoint was progression-free survival (PFS) assessed by independent central review (ICR) per RECIST 1.1 RESULTS: A total of 150 patients were randomized. The combination therapy resulted in shorter median PFS by RECIST 1.1 than axitinib monotherapy (6.7 vs. 11.4 months). The combination was tolerated similarly to axitinib monotherapy, and there were no treatment related deaths. Exploratory evaluation of pretreatment circulating biomarkers suggested the combination might benefit patients who have low baseline VEGF levels. CONCLUSION: The combination of carotuximab with axitinib did not demonstrate additional efficacy over single agent axitinib in patients with mccRCC who progressed following one or more prior VEGF inhibitor treatment.

Endoglin Targeting: Lessons Learned and Questions That Remain.

Approximately 30 years ago, endoglin was identified as a transforming growth factor (TGF)-ß coreceptor with a crucial role in developmental biology and tumor angiogenesis. Its selectively high expression on tumor vessels and its correlation with poor survival in cancer patients led to the exploration of endoglin as a therapeutic target for cancer. The endoglin neutralizing antibody TRC105 (Carotuximab®, Tracon Pharmaceuticals (San Diego, CA, USA) was subsequently tested in a wide variety of preclinical cancer models before being tested in phase I-III clinical studies in cancer patients as both a monotherapy and in combination with other chemotherapeutic and anti-angiogenic therapies. The combined data of these studies have revealed new insights into the role of endoglin in angiogenesis and its expression and functional role on other cells in the tumor microenvironment. In this review, we will summarize the preclinical work, clinical trials and biomarker studies of TRC105 and explore what these studies have enabled us to learn and what questions remain unanswered.

Endothelial damage and a thin intercellular fibrin network promote haemorrhage in acute promyelocytic leukaemia.

BACKGROUND: The role of vascular endothelium in acute promyelocytic leukaemia (APL) remains unknown. We aimed to investigate the mechanisms by which APL cells interact with endothelial cells (ECs) and to further explore how the endothelium affects bleeding as well as therapeutic interventions. METHOD: APL cells and an original APL cell line, NB4 cells, were used for experiments. The effects of leukaemic cells on ECs were analyzed in vitro and in vivo. Moreover, the endothelial barrier function and procoagulant activity were detected. An APL mouse model was established for in vivo studies. FINDINGS: APL cells interacted with ECs via ICAM-1 and VCAM-1 receptors to disrupt endothelial integrity. This binding activated MLCK signaling, resulting in the trans-endothelial passage of protein and red blood cells (RBCs). Combined treatment with asiatic acid or anti-adhesion receptor antibody inhibited the response of ECs to APL cells, thereby preventing APL-associated haemorrhage in vitro and in vivo. Activated ECs exhibited a procoagulant phenotype after phosphatidylserine exposure. Plasma from APL patients formed a thin fibrin network between procoagulant ECs, and this intercellular fibrin decreased the passage of albumin and RBCs. Ex vivo addition of fibrinogen further enhanced this barrier function in a dose-dependent manner. INTERPRETATION: Endothelial damage induced by leukaemic cell adherence promotes haemorrhaging in APL. Stabilization of ECs, decreasing adhesion receptor expression, and increasing fibrinogen transfusion levels may be a new therapeutic avenue to alleviate this fatal bleeding complication. FUNDING: National Science Foundation of China (81670128, 81873433).

Prognostic and Predictive Biomarkers in Patients with Metastatic Colorectal Cancer Receiving Regorafenib.

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 (P intx = 0.0167), VCAM-1 (P intx = 0.0216), and PDGF-AA (P intx = 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 (P intx = 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.