Simultaneous Isolation of Circulating Nucleic Acids and EV-Associated Protein Biomarkers From Unprocessed Plasma Using an AC Electrokinetics-Based Platform.

The power of personalized medicine is based on a deep understanding of cellular and molecular processes underlying disease pathogenesis. Accurately characterizing and analyzing connections between these processes is dependent on our ability to access multiple classes of biomarkers (DNA, RNA, and proteins)-ideally, in a minimally processed state. Here, we characterize a biomarker isolation platform that enables simultaneous isolation and on-chip detection of cell-free DNA (cfDNA), extracellular vesicle RNA (EV-RNA), and EV-associated proteins in unprocessed biological fluids using AC Electrokinetics (ACE). Human biofluid samples were flowed over the ACE microelectrode array (ACE chip) on the Verita platform while an electrical signal was applied, inducing a field that reversibly captured biomarkers onto the microelectrode array. Isolated cfDNA, EV-RNA, and EV-associated proteins were visualized directly on the chip using DNA and RNA specific dyes or antigen-specific, directly conjugated antibodies (CD63, TSG101, PD-L1, GPC-1), respectively. Isolated material was also eluted off the chip and analyzed downstream by multiple methods, including PCR, RT-PCR, next-generation sequencing (NGS), capillary electrophoresis, and nanoparticle size characterization. The detection workflow confirmed the capture of cfDNA, EV-RNA, and EV-associated proteins from human biofluids on the ACE chip. Tumor specific variants and the mRNAs of housekeeping gene PGK1 were detected in cfDNA and RNA isolated directly from chips in PCR, NGS, and RT-PCR assays, demonstrating that high-quality material can be isolated from donor samples using the isolation workflow. Detection of the luminal membrane protein TSG101 with antibodies depended on membrane permeabilization, consistent with the presence of vesicles on the chip. Protein, morphological, and size characterization revealed that these vesicles had the characteristics of EVs. The results demonstrated that unprocessed cfDNA, EV-RNA, and EV-associated proteins can be isolated and simultaneously fluorescently analyzed on the ACE chip. The compatibility with established downstream technologies may also allow the use of the platform as a sample preparation method for workflows that could benefit from access to unprocessed exosomal, genomic, and proteomic biomarkers.

Randomized phase 2b study of pralatrexate versus erlotinib in patients with stage IIIB/IV non-small-cell lung cancer (NSCLC) after failure of prior platinum-based therapy.

INTRODUCTION: Pralatrexate, a folate analogue targeting dihydrofolate reductase, has antitumor activity in non-small-cell lung cancer (NSCLC). This randomized phase 2b trial was designed to further evaluate pralatrexate activity in NSCLC by estimating overall survival (OS) relative to erlotinib in patients with relapsed/refractory disease. METHODS: In 43 centers across 6 countries, patients were randomized 1:1 to receive intravenous pralatrexate 190 mg/m on days 1 and 15 of a 28-day cycle, or oral erlotinib 150 mg/day. The primary objective was to estimate OS in all patients and prespecified subgroups using relative comparisons of hazard ratios (HRs). Secondary endpoints included progression-free survival, response rate, and safety. Key eligibility criteria included: (1) ≥1 prior platinum-based therapy, (2) Eastern Cooperative Oncology Group performance status of 0 to 1, and 3) a smoking history of 100 cigarettes or more. RESULTS: A total of 201 patients were randomized. A trend toward improvement in OS favoring pralatrexate was observed with an HR of 0.84 (95% confidence interval: 0.61-1.14) in the intent-to-treat population. This favorable survival result was seen in most prespecified subgroups for pralatrexate. The largest reduction in the risk of death was observed in patients with nonsquamous cell carcinoma (n = 107; HR = 0.65; 95% confidence interval: 0.42-1.0). The most common grade 3 to 4 adverse event in the pralatrexate arm was mucositis (23%). Discontinuation of pralatrexate for any grade of mucositis was 21%. CONCLUSIONS: Pralatrexate demonstrated a trend toward improved survival relative to erlotinib in patients with advanced NSCLC. Future studies should include a mucositis management plan to improve tolerability and maximize treatment benefit.

Pilot study of PD-0325901 in previously treated patients with advanced melanoma, breast cancer, and colon cancer.

PURPOSE: To assess further the tolerability and preliminary antitumor activity of PD-0325901 in previously treated patients with advanced melanoma, breast cancer, and colon cancer. METHODS: This pilot study evaluated PD-0325901 on an intermittent dosing schedule. PD-0325901 was administered orally at 20 mg twice daily (BID) for 21 consecutive days followed by 7 days of no treatment. This dose was not well tolerated and consequently changed to 15 mg BID. RESULTS: Between October and December 2005, 13 patients with metastatic measurable disease were entered into the study (seven melanoma, three breast cancer, and three colon cancer). All patients had received prior systemic therapy and were treated with a total of 61 cycles of PD-0325901 (nine received an initial dose of 20 mg BID, four an initial dose of 15 mg BID). The study was terminated early because of an unexpected high incidence of musculoskeletal and neurological adverse events, including gait disturbance, memory impairment, confusion, mental status changes, mild to moderate visual disturbances, and muscular weakness including neck weakness ("dropped-head syndrome"). Other common toxicities were diarrhea, acneiform rash, fatigue, and nausea. There was no significant hematologic toxicity, and chemistry abnormalities were rare. One patient achieved a confirmed complete response, and five patients had stable disease. CONCLUSIONS: PD-0325901 can cause significant musculoskeletal, neurological, and ocular toxicity at doses ≥ 15 mg BID. Future studies with adaptive designs might evaluate doses ≤ 10 mg BID in tumor types with a high incidence of Ras and Raf mutations. ClinicalTrials.gov identifier NCT00147550.

A randomized phase 2 study of erlotinib alone and in combination with bortezomib in previously treated advanced non-small cell lung cancer.

INTRODUCTION: This phase 2 study was conducted to determine the efficacy and safety of erlotinib alone and with bortezomib in patients with non-small cell lung cancer (NSCLC). METHODS: Patients with histologically or cytologically confirmed relapsed or refractory stage IIIb/IV NSCLC were randomized (1:1; stratified by baseline histology, smoking history, sex) to receive erlotinib 150 mg/d alone (arm A; n = 25) or in combination with bortezomib 1.6 mg/m2, days 1 and 8 (arm B; n = 25) in 21-day cycles. Responses were assessed using Response Evaluation Criteria in Solid Tumors. Tumor samples were evaluated for mutations predicting response. Six additional patients received the combination in a prior dose deescalation stage and were included in safety analyses. RESULTS: Response rates were 16% in arm A and 9% in arm B; disease control rates were 52 and 45%, respectively. The study was halted at the planned interim analysis due to insufficient clinical activity in arm B. Median progression-free survival and overall survival were 2.7 and 7.3 months in arm A, and 1.3 and 8.5 months in arm B. Six-month survival rates were 56.0% in both arms; 12-month rates were 40 and 30% in arms A and B, respectively. Response rate to erlotinib+/-bortezomib was significantly higher in patients with epidermal growth factor receptor mutations (50 versus 9% for wild type). The most common treatment-related grade > or =3 adverse event was skin rash (three patients in each treatment group). CONCLUSION: Insufficient activity was seen with erlotinib plus bortezomib in patients with relapsed/refractory advanced NSCLC to warrant a phase 3 study of the combination.

Vandetanib versus gefitinib in patients with advanced non-small-cell lung cancer: results from a two-part, double-blind, randomized phase ii study.

PURPOSE: Vandetanib is a once-daily oral inhibitor of vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) signaling. In this two-part phase II study, the efficacy and safety of vandetanib was compared with that of gefitinib, an inhibitor of EGFR signaling. PATIENTS AND METHODS: Patients (N = 168) with locally advanced or metastatic (stage IIIB/IV) non-small-cell lung cancer (NSCLC), after failure of first-line with or without second-line platinum-based chemotherapy, received once-daily vandetanib 300 mg (n = 83) or gefitinib 250 mg (n = 85) until disease progression or evidence of toxicity (part A). After a 4-week washout period, eligible patients had the option to switch to the alternative treatment (part B). Progression-free survival (PFS) was the primary efficacy assessment in part A, which was designed to have a higher than 75% power to detect a 33% prolongation of PFS at a one-sided significance level of .2. RESULTS: In part A, vandetanib prolonged PFS compared with gefitinib (hazard ratio = 0.69; 95% CI, 0.50 to 0.96; one-sided P = .013). Patients receiving vandetanib experienced adverse events that were manageable and generally consistent with inhibition of EGFR and VEGFR signaling, including diarrhea, rash, and hypertension. There were no unexpected safety findings with gefitinib. Overall survival, a secondary assessment, was not significantly different between patients initially randomly assigned to either vandetanib or gefitinib. CONCLUSION: The primary efficacy objective was achieved, with vandetanib demonstrating a significant prolongation of PFS versus gefitinib. Vandetanib 300 mg/d is currently being evaluated as a monotherapy in two randomized phase III studies in advanced NSCLC.

Randomized phase II study of gemcitabine plus cisplatin or carboplatin [corrected], with or without cetuximab, as first-line therapy for patients with advanced or metastatic non small-cell lung cancer.

PURPOSE: To evaluate the efficacy of cetuximab added to first-line gemcitabine/platinum in chemotherapy-naïve patients with advanced non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: In this noncomparative, randomized trial, chemotherapy-naïve patients with recurrent/metastatic NSCLC (stage IV or stage IIIB with malignant pleural effusion) were eligible. Patients received cisplatin (75 mg/m2 i.v., every 3 weeks) or carboplatin (area under the concentration-versus-time curve of 5 intravenously [i.v.], every 3 weeks), and gemcitabine (1,250 or 1,000 mg/m2 i.v., days 1 and 8) plus cetuximab (400 mg/m2 i.v. day 1, followed by 250 mg/m2 weekly), in arm A, or chemotherapy alone, in arm B. Response rate was the primary end point; safety, progression-free survival, and overall survival were secondary end points. RESULTS: Sixty-five patients were randomly assigned to arm A and 66 to arm B. Partial responses were observed in 18 patients (27.7%; 95% CI, 17.3 to 40.2) in arm A and 12 (18.2%; 95% CI, 9.8 to 29.6) in arm B. Median progression-free survival was 5.09 months for arm A (95% CI, 4.17 to 5.98) and 4.21 months (95% CI, 3.81 to 5.49) in arm B. Median overall survival was 11.99 months (95% CI, 8.80 to 15.18) and 9.26 months (95% CI, 7.43 to 11.79) in arms A and B, respectively. Overall toxicity was acceptable and consistent with the profiles of the individual agents. CONCLUSION: First-line treatment with cetuximab plus gemcitabine/platinum is well tolerated and can be administered safely in patients with advanced NSCLC. Differences in response rate, progression-free survival, and overall survival suggest that the addition of cetuximab to platinum/gemcitabine may improve clinical outcomes. Larger studies are in progress to address this hypothesis.

Efficacy and safety of paclitaxel poliglumex as first-line chemotherapy in patients at high risk with advanced-stage non-small-cell lung cancer: results of a phase II study.

BACKGROUND: The objectives of this open-labeled, multicenter, phase II trial were to evaluate response, survival, and tolerability in patients at high risk (Eastern Cooperative Oncology Group performance status [PS] of 2 or age >or= 70 years) with advanced-stage non-small-cell lung cancer (NSCLC) receiving single-agent paclitaxel poliglumex as first-line monotherapy. PATIENTS AND METHODS: Paclitaxel poliglumex was administered as a 10-20-minute infusion on day 1 of each 3-week cycle. Thirty patients were enrolled: 28 received paclitaxel poliglumex 175 mg/m2, and 2 received 235 mg/m2. Patients exhibiting a partial response (PR; by Response Evaluation Criteria in Solid Tumors) or stable disease (SD) continued uninterrupted treatment with paclitaxel poliglumex for or= 70 years was 7.8 months. No grade 4 nonhematologic toxicities were reported. Three patients experienced grade 3 neuropathy. Weekly hematologic assessments showed grade 3 anemia in 2 patients, grade 3 neutropenia (not associated with neutropenic fever) in 3 patients, and grade 4 neutropenia in 2 patients. No patient required growth factor support. CONCLUSION: The results of this study indicate that paclitaxel poliglumex is generally well tolerated and has activity at a dose level of 175 mg/m2 as first-line monotherapy in patients at high risk with advanced NSCLC.