Recent work identified L-asparaginase (L-ASP) as a putative therapeutic target for ovarian cancer. We hypothesized L-ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L-ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion, and the assessment of sialylated proteins involved in matrix-associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell-cell and cell-matrix adhesion was observed (p<0.05 – 0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E-selectin expression was seen consistent with the unidirectional inhibitory actions observed. L-ASP concentrations were non-toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin-1, and cleavage of LC-3, indicating cell injury did occur. These data and the known mechanism of action of L-ASP on glycosylation of nascent proteins suggest that L-ASP reduction of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behavior, and causes cell injury initiating autophagy in tumor and vascular cells.
asparaginase; ovarian cancer; sialyl Lewis X; angiogenesis; autophagy
The anti-angiogenic activity of L-asparaginase (L-ASP) and the sensitivity of ovarian cancer cell lines to L-ASP has been previously demonstrated by preclinical findings. The aim of this clinical trial was to translate those findings and evaluate the activity of polyethylene glycol-conjugated L-asparaginase (PEG-ASP or pegaspargase) in advanced ovarian cancer. Women with recurrent ovarian cancer and good end-organ function were enrolled in an open-label phase II trial of PEG-ASP at a dose of 2,000 IU/m2 by intravenous infusion every 2 weeks. Patients were evaluated for response every 8 weeks and for toxicity on an ongoing basis. Early stopping rules for toxicity and activity were included. Four patients were enrolled and received a total of 7 treatment cycles. The study ended accrual by invoking an early stopping rule, after excessive toxicity was identified in patients. Drug-related toxicities included grade 2 pancreatitis, fatigue, neutropenia, hypoalbuminemia, weight loss, dehydration, decreased fibrinogen and 1 case of grade 3 hypersensitivity reaction during cycle 2. One patient died during the study. No patients were evaluable for response. PEG-ASP was poorly tolerated in this group of advanced-stage ovarian cancer patients and no conclusions regarding activity may be drawn. Further studies of PEG-ASP in ovarian cancer patients are not recommended.
ovarian cancer; pegaspargase; L-asparaginase; angiogenesis
Recent work identified L-asparaginase (L-ASP) as a putative therapeutic target for ovarian cancer. We suggest that L-ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L-ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion and the assessment of sialylated proteins involved in matrix-associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell-cell and cell-matrix adhesion was observed (P < 0.05–0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E-selectin expression was seen consistent with the unidirectional inhibitory actions observed. L-ASP concentrations were non-toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin-1, and cleavage of LC-3, indicating cell injury did occur. These data and the known mechanism of action of L-ASP on glycosylation of nascent proteins suggest that L-ASP reduces of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behaviour, and causes cell injury initiating autophagy in tumour and vascular cells.
asparaginase; ovarian cancer; sialyl Lewis X; angiogenesis; autophagy
Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI’s invasive capacity.
Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression were graded on tissue microarray and clinical samples. Statistical comparisons used unpaired T test and ANOVA, where appropriate.
SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p<0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p<0.05) and protein (p<0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r2=0.986) and a set of ovarian cancers (p<0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p=0.04).
SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer.
ovarian cancer; SLPI; MMP-9; metalloproteinase
There are few validated relapse prediction biomarkers for epithelial ovarian cancer (EOC). We have shown progranulin (PGRN) and secretory leukocyte protease inhibitor (SLPI) are up regulated, overexpressed survival factors in EOC. We hypothesized they would predict presence of occult EOC.
PGRN, SLPI, and the known biomarker HE4 were measured in EOC patient plasma samples, prospectively collected every 3 months from initial remission until relapse. Clinical data and CA125 results were incorporated into statistical analyses. Exploratory Kaplan-Meier estimates, dividing markers at median values, evaluated association with progression-free survival (PFS) and overall survival (OS). Area-under-the-curve (AUC) statistics were computed from receiver operating characteristic (ROC) curves to evaluate discrimination ability. A Cox proportional hazards model assessed the association between PFS, OS, and biomarkers, adjusting for clinical prognostic factors.
Samples from 23 advanced stage EOC patients were evaluated. PGRN at 3 months was the only biomarker independently associated with PFS (P<0.0001) and OS (P<0.003). When used to predict progression by 18 months, sensitivity and specificity were 93% and 100%, respectively, with AUC = 0.944. The Cox model hazard ratio for PFS, divided at 59 ng/ml by ROC analysis and adjusted for clinical factors, was 23.5 (95% CI: 2.49–220). Combinations with SLPI, HE4, and/or CA125 did not improve the model.
We report pilot data indicating a potential independent association of PGRN on EOC patient PFS and OS. A validation study will be required to confirm this finding and to inform whether PGRN warrants evaluation as a potential screening biomarker.
Progranulin (PGRN); biomarkers; progression free survival; overall survival; epithelial ovarian cancer
To assess activity and toxicity in newly diagnosed advanced stage epithelial ovarian cancer (EOC) patients receiving dose-intense paclitaxel, cyclophosphamide, cisplatin, and filgrastim delivered with a flexible dosing schedule.
Patients with Stage III/IV EOC received cyclophosphamide 750 mg/m2, followed by 24 hr infusion of paclitaxel 250 mg/m2, and cisplatin 75 mg/m2 on day 2. Filgrastim began on day 3 at 10 μg/kg/d × 9d. Patients received six cycles of all drugs. Those with pathologic complete response or microscopic residual disease at the conclusion of six cycles of therapy received an additional cycles two to four cycles of paclitaxel with cyclophosphamide. Patients with objective response continued cyclophosphamide and paclitaxel.
62 patients were enrolled. Thirty-two of these 62 patients had stage IIIC disease, and 26 of 62 had stage IV disease. Using an intent to treat analysis, 55 (89%) experienced clinical complete remission (CCR). With a median potential follow-up of 11.4 years, the median progression free survival is 18.9 months and median survival is 5.4 years. The most serious toxicity was grade 3/4 neutropenic fever (35%). Although all participants developed peripheral neuropathy, improvement in neuropathic symptoms began with decrease or cessation of paclitaxel.
This regimen yielded a high response rate and encouraging overall survival. These data and those of the Japanese Gynecologic Oncology Group suggest that further study of dose dense or intense paclitaxel regimens in women with newly diagnosed advanced stage EOC is warranted.
ovarian neoplasms; paclitaxel; cyclophosphamide; cisplatin; antineoplastic combined chemotherapy protocols; filgrastim; drug dose-response relationship
Ovarian cancer (OvCa) recurrence with development of paclitaxel resistance is an obstacle to long term survival. We demonstrated that secretory leukocyte protease inhibitor (SLPI) is a survival factor for OvCa. We hypothesize SLPI may antagonize paclitaxel injury.
Differential SLPI induction in response to paclitaxel, and response to stable forced expression of SLPI was demonstrated in A2780-1A9 cells and their paclitaxel-resistant sublines, PTX10 and PTX22 and confirmed with HEY-A8 cells. SLPI-mediated survival was reduced by the MEK inhibitor, U0126 and a humanized neutralizing monoclonal anti-SLPI antibody, CR012. OVCAR3 xenographs tested the role of CR012 in vivo.
SLPI expression was lower in A2780-1A9 OvCa cells than PTX10 and PTX22 and SLPI was induced by paclitaxel exposure. Stable SLPI expression yielded a proliferation advantage (p=0.01); expression of and response to SLPI in OVCAR3 cells was abrogated by exposure to CR012. SLPI reduced paclitaxel susceptibility of 1A9 and HEY-A8 cells (p≤0.05) and SLPI expression did not increase resistance of PTX10 and −22 cells. Both paclitaxel and SLPI overexpression induced ERK activation. Inhibition of MEK with U0126 increased paclitaxel injury and overcame SLPI-mediated cell protection. It did not reinstate PTX10 sensitivity to paclitaxel, which was associated with AKT activation. Significant inhibition of OVCAR3 xenograft growth was observed with CR012 and paclitaxel, over single agents (p≤0.001).
A two-pronged approach confirmed SLPI overcomes paclitaxel in part through activation of ERK1/2. These results credential SLPI as a molecular target for OvCa and suggest CR012 as a tool for proof of concept.
Secretory leukocyte protease inhibitor (SLPI); ovarian cancer; paclitaxel; resistance
Novel technologies are now being advanced for the purpose of identification and validation of new disease biomarkers. A reliable and useful clinical biomarker must a) come from a readily attainable source, such as blood or urine, b) have sufficient sensitivity to correctly identify affected individuals, c) have sufficient specificity to avoid incorrect labeling of unaffected persons, and d) result in a notable benefit for the patient through intervention, such as survival or life quality improvement. Despite these critical descriptors, the few available FDA-approved biomarkers for cancer do not completely fit this definition and their benefits are limited to a small number of cancers. Ovarian cancer exemplifies the need for a diagnostic biomarker of early stage disease. Symptoms are present but not specific to the disease, delaying diagnosis until an advanced and generally incurable stage in over 70% of affected women. As such, diagnostic intervention in the form of oopherectomy can be performed in the appropriate at-risk population if identified such as with a new accurate, sensitive, and specific biomarker. If early stage disease is identified, the requirement for survival and life quality improvement will be met. One of the new technologies applied to biomarker discovery is tour-de-force analysis of serum peptides and proteins. Optimization of mass spectrometry techniques coupled with advanced bioinformatics approaches has yielded informative biomarker signatures discriminating presence of cancer from unaffected in multiple studies from different groups. Validation and randomized outcome studies are needed to determine the true value of these new biomarkers in early diagnosis, and improved survival and quality of life.
Ovarian cancer; proteomics; mass spectrometry; biomarker; diagnosis
PARP inhibitors (PARPi) are a novel class of drugs with activity in patients with acquired or germline homologous recombination (HR) deficiency-associated high-grade serous ovarian cancer (HGSOC). We hypothesized that measuring γH2AX as an indicator of DNA double-strand breaks (DSB), and MRE11 or RAD51 as an indicator of DSB repair, would reflect HR status and predict response to PARPi-based therapy. Our aim was to develop and use high-throughput multiparametric flow cytometry to quantify γH2AX with MRE11 or RAD51 in PBMCs as a readily available surrogate.
Healthy donor PBMCs were used for assay development and optimization. We validated induction of γH2AX, MRE11 and RAD51 by staining with fluorophore-conjugated antibodies. The multiparameter flow cytometric method was applied to PBMC samples from recurrent HGSOC patients who were treated with PARPi, olaparib and carboplatin.
Stimulation was necessary for quantification of a DNA damage response to olaparib/carboplatin in healthy donor PBMCs. The flow cytometric protocol could not distinguish between cytoplasmic and nuclear RAD51, erroneously indicating activation in response to injury. Thus, MRE11 was selected as the marker of DSB repair. PBMCs from 15 recurrent HGSOC patients were then examined. Patients who did not respond to PARPi therapy had a significantly higher pre-treatment level of γH2AX (p = 0.01), and a higher ratio of γH2AX/MRE11 (11.0 [3.5–13.2] v. 3.3 [2.8–9.9], p < 0.03) compared with responders.
We successfully developed and applied a multiparameter flow cytometry assay to measure γH2AX and MRE11 in PBMCs. Prospective studies will be required to validate this surrogate biomarker assay as a potential predictive biomarker of PARPi-based therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0604-z) contains supplementary material, which is available to authorized users.
Ovarian cancer; PARP inhibitor; Biomarkers; Flow cytometry; Peripheral blood mononuclear cells; γH2AX; MRE11; RAD51
Olaparib has single-agent activity against breast/ovarian cancer (BrCa/OvCa) in germline BRCA1 or BRCA2 mutation carriers (gBRCAm). We hypothesized addition of olaparib to carboplatin can be administered safely and yield preliminary clinical activity.
Eligible patients had measurable or evaluable disease, gBRCAm, and good end-organ function. A 3 + 3 dose escalation tested daily oral capsule olaparib (100 or 200mg every 12 hours; dose level1 or 2) with carboplatin area under the curve (AUC) on day 8 (AUC3 day 8), then every 21 days. For dose levels 3 to 6, patients were given olaparib days 1 to 7 at 200 and 400 mg every 12 hours, with carboplatin AUC3 to 5 on day 1 or 2 every 21 days; a maximum of eight combination cycles were permitted, after which daily maintenance of olaparib 400mg every12 hours continued until progression. Dose-limiting toxicity was defined in the first two cycles. Peripheral blood mononuclear cells were collected for polymorphism analysis and polyADP-ribose incorporation. Paired tumor biopsies (before/after cycle 1) were obtained for biomarker proteomics and apoptosis endpoints.
Forty-five women (37 OvCa/8 BrCa) were treated. Dose-limiting toxicity was not reached on the intermittent schedule. Expansion proceeded with olaparib 400mg every 12 hours on days 1 to 7/carboplatin AUC5. Grade 3/4 adverse events included neutropenia (42.2%), thrombocytopenia (20.0%), and anemia (15.6%). Responses included 1 complete response (1 BrCa; 23 months) and 21 partial responses (50.0%; 15 OvCa; 6 BrCa; median = 16 [4 to >45] in OvCa and 10 [6 to >40] months in BrCa). Proteomic analysis suggests high pretreatment pS209-eIF4E and FOXO3a correlated with duration of response (two-sided P < .001; Pearson’s R
2 = 0.94).
Olaparib capsules 400mg every 12 hours on days 1 to 7/carboplatin AUC5 is safe and has activity in gBRCAm BrCa/OvCa patients. Exploratory translational studies indicate pretreatment tissue FOXO3a expression may be predictive for response to therapy, requiring prospective validation.
The Ovarian Task Force of the Gynecologic Cancer Steering Committee convened a clinical trials planning meeting on October 28–29, 2011, with the goals to identify key tumor types, associated molecular pathways, and biomarkers for targeted drug intervention; review strategies to improve early-phase screening, therapeutic evaluation, and comparison of new agents; and optimize design of randomized trials in response to an evolving landscape of scientific, regulatory, and funding priorities. The meeting was attended by international clinical and translational investigators, pharmaceutical industry representatives, government regulators, and patient advocates. Panel discussions focused on disease types, early-phase trials, and randomized trials. A manuscript team summarized the discussions and assisted with formulating key recommendations. A more integrated and efficient approach for screening new agents using smaller selective randomized trials in specific disease-type settings was endorsed, together with collaborative funding models between industry and the evolving national clinical trials network, as well as efforts to enhance public awareness and study enrollment through advocacy.
Olaparib (O), a polyADPribose polymerase (PARP) inhibitor, and cediranib (C), a VEGF receptor (VEGFR)1–3 inhibitor together had greater activity than O alone in women with recurrent platinum-sensitive ovarian cancer (OvCa). The objective of this study is to identify potential lead biomarker candidates for response to O + C in the setting of a multi-institutional phase II study of O with and without C in recurrent platinum-sensitive OvCa.
A self-selected group of patients participated in a prospectively planned exploratory biomarker substudy of the randomized phase II study of O versus O + C. Whole blood for peripheral blood mononuclear cell (PBMC) and plasma isolation was collected prior to and on day 3 of treatment. Quantitation of circulating endothelial cells (CEC), IL-6, IL-8, VEGF, and soluble VEGFR-2 plasma concentrations, and polyADPribose (PAR) incorporation were performed. Single nucleotide polymorphism analysis of XRCC1 280H, R194W, and Q399R was done. Dynamic contrast-enhanced-magnetic resonance imaging (DCE-MRI) was performed at baseline and day 3 of treatment. Parameter changes were compared between the two arms using an exact Wilcoxon rank sum test. Kaplan–Meier and log-rank tests were used to examine survival outcome.
Thirteen patients elected to participate in the translational substudy, seven patients on O and six patients on O + C. Patients on O + C had a greater decrease in IL-8 concentration and larger CEC fold increase compared with those on O alone (p = 0.026, p = 0.032). The fold increase in CEC on day 3 was associated with duration of progression-free survival (PFS) (R2 = 0.77, 95% CI 0.55–0.97, p < 0.001). IL-8 post-pretreatment changes correlate with PFS (p = 0.028). XRCC1 DNA polymorphisms were not related to PFS. All patients had reduction in PAR incorporation, and all except one had reduction in vascular flow on DCE-MRI.
Our exploratory correlative studies indicate that CEC and IL-8 changes may be predictive for response to O + C and prognostic in recurrent platinum-sensitive OvCa, requiring prospective validation.
CEC; IL-8; biomarkers; olaparib; cediranib; ovarian cancer
We recently used RNAi to demonstrate that a negative correlation of L-asparaginase (L-ASP) chemotherapeutic activity with asparagine synthetase (ASNS) expression in the ovarian subset of the NCI-60 cell line panel is causal. To determine whether that relationship would be sustained in a larger, more diverse set of ovarian cell lines, we have now measured ASNS mRNA expression using microarrays and a branched-DNA RNA assay, ASNS protein expression using an electrochemiluminescent immunoassay, and L-ASP activity using an MTS assay on nineteen human ovarian cancer cell lines. Contrary to our previous findings, L-ASP activity was only weakly correlated with ASNS mRNA expression; Pearson’s correlation coefficients were r = −0.21 for microarray data and r = −0.39 for the branched-DNA RNA assay, with just the latter being marginally statistically significant (p = 0.047, one-tailed). ASNS protein expression measured by liquid phase immunoassay exhibited a much stronger correlation, r = −0.65 (p = 0.0014, one-tailed). We conclude that ASNS protein expression measured by immunoassay is a strong univariate predictor of L-ASP activity in ovarian cancer cell lines. These findings provide rationale for clinical evaluation of ASNS protein expression as a predictive biomarker of L-ASP activity in ovarian cancer.
asparagine synthetase; asparaginase; ovarian cancer; biomarker; pharmacogenomics
As personalized medicine becomes more applicable to oncologic practice, image-guided biopsies will be integral for enabling predictive and pharmacodynamic molecular pathology. Interventional radiology has a key role in defining patient-specific management. Advances in diagnostic techniques, genomics, and proteomics enable a window into subcellular mechanisms driving hyperproliferation, metastatic capabilities, and tumor angiogenesis. A new era of personalized medicine has evolved whereby clinical decisions are adjusted according to a patient’s molecular profile. Several mutations and key markers already have been introduced into standard oncologic practice. A broader understanding of personalized oncology will help interventionalists play a greater role in therapy selection and discovery.
There has been increasing interest in serial research biopsies in studies of targeted therapies. Definition of patient characteristics and optimal target tissue for safe research tumor biopsy in the era of anti-angiogenic and targeted agents is needed.
This IRB-approved retrospective study included chart and interventional radiology case review from six phase 1/2 studies at the NCI.
142 of 150 protocol patients approached gave consent for research biopsies. Patients had a median age of 56 yrs (27–78), median BMI 25.8 kg/m2 (14.4–46.2), ECOG PS 0–1, and normal end-organ function. Baseline biopsies were collected in 138/142 patients (97%), and paired specimens in 96(70%). Most patients had metastatic gynecologic cancers (85%) and 78% patients had target disease below the diaphragm of median size 2.7cm (1–14.5cm). Protocol therapies included kinase inhibitors (35%), angiogenesis inhibitors (54%), and olaparib/carboplatin (11%); therapy was not interrupted for biopsies. Adverse events were all uncomplicated and were observed in four patients (liver subcapsular hematoma ; vasovagal syncope ; pneumothorax ). The complication rate in obese patients was similar to that in non-obese patients (3/108 vs.1/34). 67 patients (48%) were receiving bevacizumab at the time of subsequent biopsies. The complication rate in those receiving bevacizumab was not different from those without (3/67 vs 1/71). 95% of biopsies yielded useable material.
Serial percutaneous core needle biopsies can be obtained safely and yield material applicable for multiple translational applications. Obesity and/or concomitant anti-angiogenic therapy, and depth of disease do not increase risk or preclude successful acquisition of useful tissue.
Epithelial ovarian cancer, once categorizing all epithelial cancers of the ovary and fallopian tube, is now recognized to be an umbrella term. We are recognizing two categories of ovarian cancer, with the “type 1” cancers containing further types, including low grade serous cancers, mucinous, clear cell, and low grade endometrioid. These types are genetically and histologically as different as is their outcome. The paper accompanying this editorial further dissects low grade serous cancers to show that those carrying oncogenic KRAS or BRAF mutations have an unexpectedly excellent clinical outcome. We discuss this newest unexpected behavior of ovarian cancer.
ovarian cancer; KRAS; BRAF; serous; borderline tumor
Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown clinical activity in patients with germline BRCA1/2 mutation (gBRCAm)-associated breast and ovarian cancers. Accumulating evidence suggests that PARPi may have a wider application in the treatment of cancers defective in DNA damage repair pathways, such as prostate, lung, endometrial, and pancreatic cancers. Several PARPi are currently in phase I/II clinical investigation, as single-agents and/or combination therapy in these solid tumors. Understanding more about the molecular abnormalities involved in BRCA-like phenotype in solid tumors beyond breast and ovarian cancers, exploring novel therapeutic trial strategies and drug combinations, and defining potential predictive biomarkers are critical to expanding the scope of PARPi therapy. This will improve clinical outcome in advanced solid tumors. Here, we briefly review the preclinical data and clinical development of PARPi, and discuss its future development in solid tumors beyond gBRCAm-associated breast and ovarian cancers.
poly(ADP-ribose) polymerase inhibitors; solid tumors; BRCA mutation; BRCA-like; DNA damage repair pathway
The present study is designed to assess if exosomes released from Chronic Myelogenous Leukemia (CML) cells may modulate angiogenesis. We have isolated and characterized the exosomes generated from LAMA84 CML cells and demonstrated that addition of exosomes to human vascular endothelial cells (HUVEC) induces an increase of both ICAM-1 and VCAM-1 cell adhesion molecules and interleukin-8 expression. The stimulation of cell-cell adhesion molecules was paralleled by a dose-dependent increase of adhesion of CML cells to a HUVEC monolayer. We further showed that the treatment with exosomes from CML cells caused an increase in endothelial cell motility accompanied by a loss of VE-cadherin and β-catenin from the endothelial cell surface. Functional characterization of exosomes isolated from CML patients confirmed the data obtained with exosomes derived from CML cell line. CML exosomes caused reorganization into tubes of HUVEC cells cultured on Matrigel. When added to Matrigel plugs in vivo, exosomes induced ingrowth of murine endothelial cells and vascularization of the Matrigel plugs. Our results suggest for the first time that exosomes released from CML cells directly affect endothelial cells modulating the process of neovascularization.
Exosomes; Chronic Myelogenous Leukemia Cells; Endothelial cells; Tumor Microenvironment
Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length (P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release (P <0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes (P <0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment (P <0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment.
Exosomes; Nanotubes; Chronic myeloid leukemia; Endothelial cells; Tyrosine kinase inhibitors
Proteomics allows characterization of protein structure and function, protein-protein interactions, and peptide modifications. It has given us insight into the perturbations of signaling pathways within tumor cells and has improved the discovery of new therapeutic targets and possible indicators of response to and duration of therapy. The discovery, verification, and validation of novel biomarkers are critical in streamlining clinical development of targeted compounds, and directing rational treatments for patients whose tumors are dependent upon select signaling pathways. Studies are now underway in many diseases to examine the immune or inflammatory proteome, vascular proteome, cancer or disease proteome, and other subsets of the specific pathology microenvironment. Successful assay verification and biological validation of such biomarkers will speed development of potential agents to targetable dominant pathways and lead to selection of individuals most likely to benefit. Reconsideration of analytical and clinical trials methods for acquisition, examination, and translation of proteomics data must occur before we march further into future of drug development.
proteomics; biomarkers; clinical trial; drug development; cancer; targeted therapy
To characterize the pharmacokinetics (PK) of sorafenib in patients with solid tumours and to evaluate the possible effects of demographic, clinical and pharmacogenetic (CYP3A4*1B, CYP3A5*3C, UGT1A9*3 and UGT1A9*5) covariates on the disposition of sorafenib.
PK were assessed in 111 patients enrolled in five phase I and II clinical trials, where sorafenib 200 or 400 mg was administered twice daily as a single agent or in combination therapy. All patients were genotyped for polymorphisms in metabolic enzymes for sorafenib. Population PK analysis was performed by using nonlinear mixed effects modelling (NONMEM). The final model was validated using visual predictive checks and nonparametric bootstrap analysis.
A one compartment model with four transit absorption compartments and enterohepatic circulation (EHC) adequately described sorafenib disposition. Baseline bodyweight was a statistically significant covariate for distributional volume, accounting for 4% of inter-individual variability (IIV). PK model parameter estimates (range) for an 80 kg patient were clearance 8.13 l h−1 (3.6–22.3 l h−1), volume 213 l (50–1000 l), mean absorption transit time 1.98 h (0.5–13 h), fraction undergoing EHC 50% and average time to gall bladder emptying 6.13 h.
Overall, population PK analysis was consistent with known biopharmaceutical/PK characteristics of oral sorafenib. No clinically important PK covariates were identified.
CYP3A4; population pharmacokinetics; sorafenib; tyrosine kinase inhibitor; UGT1A9
To compare the recurrence-free interval (RFI), and safety profile in patients with completely resected high-risk early-stage ovarian cancer patients treated with intravenous (IV) carboplatin and paclitaxel with or without maintenance low-dose paclitaxel for 24 weeks.
Eligibility was limited to patients with Stage I-A/B (Grade 3 or clear cell), all I-C or II epithelial ovarian cancer. All patients were to receive carboplatin AUC 6 and paclitaxel 175 mg/m2 q 3 wks × 3 courses with random assignment to either observation or maintenance paclitaxel 40 mg/m2/wk × 24 wks. Recurrence required clinical or radiological evidence of new tumor.
There were 571 patients enrolled onto this study, of whom 29 were deemed ineligible due to inappropriate stage or pathology, leaving 542 patients. At least 3 cycles of treatment were administered to 524/542 (97%) of patients, and among those assigned to maintenance paclitaxel, 80% completed the regimen. The incidence of grade 2 or worse peripheral neuropathy (15.5% vs 6%), infection/fever (19.9% vs 8.7%), and dermatologic events (70.8% vs 52.1%) were higher on the maintenance regimen (p<0.001). The cumulative probability of recurring within 5 years for the maintenance paclitaxel regimen is 20% vs. 23% for surveillance (hazard ratio 0.807; 95% CI: 0.565–1.15). The probability of surviving 5 years was 85.4% and 86.2%, respectively.
Maintenance paclitaxel at 40 mg/m2/wk × 24 wks added to standard dose AUC6 and paclitaxel 175 mg/m2 × 3 doses provides no significant increase in RFI.
Background Targeting the cell-surface receptor EphA2, which is highly expressed in some solid tumors, is a novel approach for cancer therapy. We aimed to evaluate the safety profile, maximum tolerated dose (MTD), pharmacokinetics, and antitumor activity of MEDI-547, an antibody drug conjugate composed of the cytotoxic drug auristatin (toxin) linked to a human anti-EphA2 monoclonal antibody (1C1), in patients with solid tumors relapsed/refractory to standard therapy. Methods In this phase 1, open-label study with planned dose-escalation and dose-expansion cohorts, patients received a 1-h intravenous infusion of MEDI-547 (0.08 mg/kg) every 3 weeks. Results Six patients received 0.08 mg/kg; all discontinued treatment. Dose escalation was not pursued. The study was stopped before cohort 2 enrollment due to treatment-related bleeding and coagulation events (hemorrhage-related, n = 3; epistaxis, n = 2). Therefore, lower doses were not explored and an MTD could not be selected. The most frequently reported treatment-related adverse events (AEs) were increased liver enzymes, decreased hemoglobin, decreased appetite, and epistaxis. Three patients (50%) experienced treatment-related serious AEs, including conjunctival hemorrhage, pain (led to study drug discontinuation), liver disorder, and hemorrhage. Best response included progressive disease (n = 5; 83.3%) and stable disease (n = 1; 16.7%). Minimal or no dissociation of toxin from 1C1 conjugate occurred in the blood. Serum MEDI-547 concentrations decreased rapidly, ~70% by 3 days post-dose. No accumulation of MEDI-547 was observed at 0.08 mg/kg upon administration of a second dose 3 weeks following dose 1. Conclusions The safety profile of MEDI-547 does not support further clinical investigation in patients with advanced solid tumors.
MEDI-547; EphA2; Cancer therapy; Clinical trial; Relapsed/refractory solid tumors
The power of proteomics allows unparalleled opportunity to query the molecular mechanisms of a malignant cell and the tumor microenvironment in patients with ovarian cancer and other solid tumors. This information has given us insight into the perturbations of signaling pathways within tumor cells and has aided the discovery of new drug targets for the tumor and possible prognostic indicators of outcome and disease response to therapy. Proteomics analysis of serum and ascites has also given us sources with which to discover possible early markers for the presence of new disease and for the progression of established cancer throughout the course of treatment. Unfortunately, this wealth of information has yielded little to date in changing the clinical care of these patients from a diagnostic, prognostic, or treatment perspective. The rational examination and translation of proteomics data in the context of past clinical trials and the design of future clinical trials must occur before we can march forward into the future of personalized medicine.
ovarian cancer; proteomics; clinical trials
Recent work suggested a role for NF-kB in the propagation of ovarian cancer cell lines, but the significance and mechanism of NF-kB in ovarian cancer is unknown. We hypothesized that the NF-kB pathway is over-activated in aggressive ovarian cancers.
We assessed the levels of three NF-kB transcription factors, the activating IkB kinases and the NF-kB target MMP-9 by immunohistochemistry in ovarian cancer specimens obtained at diagnosis from a cohort of 33 patients subsequently treated with paclitaxel, cisplatin, and cyclophosphamide. Associations were made between NF-kB pathway proteins and outcome. Validation of co-expression was performed at the gene level in two independently collected cohorts of 185 and 153 ovarian cancers, respectively.
We established the presence of NF-kB proteins in newly diagnosed advanced ovarian cancers, and identified a potential association with overall survival. Transcription factors p65 and RelB were co-expressed with IKKα, one component of a key tri-molecular regulatory complex. Co-expression of the NF-kB machinery suggests activity of NF-kB signaling in these ovarian tumors. A significant association of p50 with poor overall survival was found (p=0.02). MMP9 expression showed the opposite relationship, where cases without MMP9 staining had the poorest prognosis (p=0.01), and this relationship held true at the gene expression level in an independently collected cohort of 185 ovarian cancers.
Deregulation of NF-κB activity may influence outcome in women treated with standard therapy for advanced ovarian cancer. Modification of the pathway could present an opportunity to improve outcome in the subset of women showing activity of the pathway.
NF-κB; ovarian cancer; immunohistochemistry; survival; p50; MMP9; prognosis