Analytical performance of the FDA-cleared Parsortix® PC1 system.

Introduction: The Parsortix® PC1 system, Food and Drug Administration (FDA) cleared for use in metastatic breast cancer (MBC) patients, is an epitope-independent microfluidic device for the capture and harvest of circulating tumor cells from whole blood based on cell size and deformability. This report details the analytical characterization of linearity, detection limit, precision, and reproducibility for this device. Methods: System performance was determined using K2-EDTA blood samples collected from self-declared healthy female volunteers (HVs) and MBC patients spiked with prelabeled cultured breast cancer cell lines (SKBR3, MCF7, or Hs578T). Samples were processed on Parsortix® PC1 systems and captured cells were harvested and enumerated. Results: The system captured and harvested live SKBR3, MCF7, and Hs578T cells and fixed SKBR3 cells linearly between 2 and ~100 cells, with average harvest rates of 69%, 73%, 79%, and 90%, respectively. To harvest ≥1 cell ≥95% of the time, the system required 3, 5 or 4 live SKBR3, MCF7 or Hs578T cells, respectively. Average harvest rates from precision studies using 5, 10, and ~50 live cells spiked into blood for each cell line ranged from 63.5% to 76.2%, with repeatability and reproducibility percent coefficient of variation (%CV) estimates ranging from 12.3% to 32.4% and 13.3% to 34.1%, respectively. Average harvest rates using ~20 fixed SKBR3 cells spiked into HV and MBC patient blood samples were 75.0% ± 16.1% (%CV = 22.3%) and 68.4% ± 14.3% (%CV = 21.1%), respectively. Conclusions: These evaluations demonstrate the Parsortix® PC1 system linearly and reproducibly harvests tumor cells from blood over a range of 1 to ~100 cells.

Specific loss of adipocyte CD248 improves metabolic health via reduced white adipose tissue hypoxia, fibrosis and inflammation.

BACKGROUND: A positive energy balance promotes white adipose tissue (WAT) expansion which is characterized by activation of a repertoire of events including hypoxia, inflammation and extracellular matrix remodelling. The transmembrane glycoprotein CD248 has been implicated in all these processes in different malignant and inflammatory diseases but its potential impact in WAT and metabolic disease has not been explored. METHODS: The role of CD248 in adipocyte function and glucose metabolism was evaluated by omics analyses in human WAT, gene knockdowns in human in vitro differentiated adipocytes and by adipocyte-specific and inducible Cd248 gene knockout studies in mice. FINDINGS: CD248 is upregulated in white but not brown adipose tissue of obese and insulin-resistant individuals. Gene ontology analyses showed that CD248 expression associated positively with pro-inflammatory/pro-fibrotic pathways. By combining data from several human cohorts with gene knockdown experiments in human adipocytes, our results indicate that CD248 acts as a microenvironmental sensor which mediates part of the adipose tissue response to hypoxia and is specifically perturbed in white adipocytes in the obese state. Adipocyte-specific and inducible Cd248 knockouts in mice, both before and after diet-induced obesity and insulin resistance/glucose intolerance, resulted in increased microvascular density as well as attenuated hypoxia, inflammation and fibrosis without affecting fat cell volume. This was accompanied by significant improvements in insulin sensitivity and glucose tolerance. INTERPRETATION: CD248 exerts detrimental effects on WAT phenotype and systemic glucose homeostasis which may be reversed by suppression of adipocyte CD248. Therefore, CD248 may constitute a target to treat obesity-associated co-morbidities.

The Parsortix™ Cell Separation System-A versatile liquid biopsy platform.

Cancer cells from solid tumors can enter the circulatory system and survive to subsequently form distant metastases. The CellSearch® system (Menarini-Silicon Biosystems, Huntingdon Valley, PA) was the first, FDA-cleared system that provided a reliable tool for the investigation of circulating tumor cells (CTCs), which have been shown to be strongly associated with poor survival and therapy failure. Since that time, a number of new technologies have been introduced to improve CTC detection and/or isolation for further characterization. The continued and growing interest in the "liquid biopsy" field has spurred the development of numerous different CTC technologies. However, selecting the most appropriate CTC platform for individual applications can be challenging. No consensus has yet been reached in the community regarding which liquid biopsy technology is optimal. Here, we introduce the Parsortix™ Cell Separation System (ANGLE North America, Inc., King of Prussia, PA), a microfluidic based technology that captures rare cells based on size and deformability, offers reproducibly high capture efficiency, and produces highly enriched, viable (viability dependent on preservative used) CTCs that are amenable to a multitude of downstream analyses, including the isolation and interrogation of single cells. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

A phase 2 study of ontuxizumab, a monoclonal antibody targeting endosialin, in metastatic melanoma.

Objectives Ontuxizumab (MORAB-004) is a first-in-class monoclonal antibody that interferes with endosialin function, which is important in tumor stromal cell function, angiogenesis, and tumor growth. This Phase 2 study evaluated the 24-week progression-free survival (PFS) value, pharmacokinetics, and tolerability of 2 doses of ontuxizumab in patients with metastatic melanoma. Patients and methods Patients with metastatic melanoma and disease progression after receiving at least 1 prior systemic treatment were randomized to receive ontuxizumab (2 or 4 mg/kg) weekly, without dose change, until disease progression. Results Seventy-six patients received at least 1 dose of ontuxizumab (40 received 2 mg/kg, 36 received 4 mg/kg). The primary endpoint, 24-week PFS value, was 11.4% (95% Confidence Interval [CI]: 5.3%-19.9%) for all patients (13.5% for 2 mg/kg and 8.9% for 4 mg/kg). The median PFS for all patients was 8.3 weeks (95% CI: 8.1-12.3 weeks). One patient receiving 4 mg/kg had a partial response, as measured by Response Evaluation Criteria in Solid Tumors v1.1. Twenty-seven of 66 response evaluable patients (40.9%) had stable disease. The median overall survival was 31.0 weeks (95% CI: 28.3-44.0 weeks). The most common adverse events overall were headache (55.3%), fatigue (48.7%), chills (42.1%), and nausea (36.8%), mostly grade 1 or 2. Conclusions Ontuxizumab at both doses was well tolerated. The 24-week PFS value was 11.4% among all ontuxizumab-treated patients. The overall response rate was 3.1% at the 4 mg/kg dose, with clinical benefit achieved in 42.4% of response evaluable patients. Efficacy of single-agent ontuxizumab at these doses in melanoma was low.

A Randomized, Double-Blind, Placebo-Controlled Phase II Study of the Efficacy and Safety of Monotherapy Ontuxizumab (MORAb-004) Plus Best Supportive Care in Patients with Chemorefractory Metastatic Colorectal Cancer.

Purpose: The purpose of this study was to evaluate the safety and efficacy of ontuxizumab (MORAb-004), a monoclonal antibody that interferes with endosialin (tumor endothelial marker-1) function, in patients with chemorefractory metastatic colorectal cancer and to identify a responsive patient population based on biomarkers.Experimental Design: This was a randomized, double-blind, placebo-controlled, phase II study. Patients were randomly assigned in a 2:1 ratio to receive weekly intravenous ontuxizumab (8 mg/kg) or placebo plus best supportive care until progression or unacceptable toxicity. Tissue and blood biomarkers were evaluated for their ability to identify a patient population that was responsive to ontuxizumab.Results: A total of 126 patients were enrolled. No significant difference between the ontuxizumab and placebo groups was evident for the primary endpoint of progression-free survival (PFS), with a median PFS of 8.1 weeks in each group (HR, 1.13; 95% confidence interval, 0.76-1.67; P = 0.53). There were no significant differences between groups for overall survival (OS) or overall response rate (ORR). The most common treatment-emergent adverse events (TEAEs) in the ontuxizumab group (vs. the placebo group, respectively) were fatigue (53.7% vs. 47.5%), nausea (39.0% vs. 35.0%), decreased appetite (34.1% vs. 27.5%), and constipation (28.0% vs. 32.5%). The most common grade 3/4 TEAE in the ontuxizumab group versus placebo was back pain (11.0% vs. 0%). No single biomarker clearly identified patients responsive to ontuxizumab.Conclusions: No benefit with ontuxizumab monotherapy compared with placebo for clinical response parameters of PFS, OS, or ORR was demonstrated. Ontuxizumab was well tolerated. Clin Cancer Res; 24(2); 316-25. ©2017 AACR.

FCGR2A and FCGR3A Genotypes Correlate with Farletuzumab Response in Patients with First-Relapsed Ovarian Cancer Exhibiting Low CA125.

Farletuzumab is a humanized monoclonal antibody that binds to folate receptor alpha and elicits an anti-tumor response via immune effector activity. Recent studies from a global phase 3 trial in ovarian cancer patients treated with carboplatin/taxane plus farletuzumab found that the tumor-produced CA125 protein can suppress farletuzumab function via perturbing its engagement to the activating Fc-γ receptors CD32a (FCGR2A) and CD16a (FCGR3A). Previous reports have indicated that naturally occurring polymorphisms in both of these receptors may play a role in their ability to engage therapeutic antibodies and elicit an optimal immune response via antibody-dependent cellular cytotoxicity (ADCC). In light of the importance of farletuzumab ADCC function for optimal tumor cell killing, we evaluated the frequency of FCGR2A-131H/R and FCGR3A-158V/F polymorphisms in 461 consenting patients from this global clinical study and their association with clinical outcome to placebo versus farletuzumab treatment. Here, we show that farletuzumab has enhanced binding to FCGR3A-158V high-affinity receptor and has an enhanced clinical outcome in patients with low baseline CA125 levels and at least 1 high-affinity allele of FCGR2A or FCGR3A.

Correlation of FCGRT genomic structure with serum immunoglobulin, albumin and farletuzumab pharmacokinetics in patients with first relapsed ovarian cancer.

Farletuzumab (FAR) is a humanized monoclonal antibody (mAb) that binds to folate receptor alpha. A Ph3 trial in ovarian cancer patients treated with carboplatin/taxane plus FAR or placebo did not meet the primary statistical endpoint. Subgroup analysis demonstrated that subjects with high FAR exposure levels (Cmin>57.6µg/mL) showed statistically significant improvements in PFS and OS. The neonatal Fc receptor (fcgrt) plays a central role in albumin/IgG stasis and mAb pharmacokinetics (PK). Here we evaluated fcgrt sequence and association of its promoter variable number tandem repeats (VNTR) and coding single nucleotide variants (SNV) with albumin/IgG levels and FAR PK in the Ph3 patients. A statistical correlation existed between high FAR Cmin and AUC in patients with the highest quartile of albumin and lowest quartile of IgG1. Analysis of fcgrt identified 5 different VNTRs in the promoter region and 9 SNVs within the coding region, 4 which are novel.

Novel antibody probes for the characterization of endosialin/TEM-1.

Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.

Endosialin and Associated Protein Expression in Soft Tissue Sarcomas: A Potential Target for Anti-Endosialin Therapeutic Strategies.

Endosialin (CD248, TEM-1) is expressed in pericytes, tumor vasculature, tumor fibroblasts, and some tumor cells, including sarcomas, with limited normal tissue expression, and appears to play a key role in tumor-stromal interactions, including angiogenesis. Monoclonal antibodies targeting endosialin have entered clinical trials, including soft tissue sarcomas. We evaluated a cohort of 94 soft tissue sarcoma samples to assess the correlation between gene expression and protein expression by immunohistochemistry for endosialin and PDGFR-ß, a reported interacting protein, across available diagnoses. Correlations between the expression of endosialin and 13 other genes of interest were also examined. Within cohorts of soft tissue diagnoses assembled by tissue type (liposarcoma, leiomyosarcoma, undifferentiated sarcoma, and other), endosialin expression was significantly correlated with a better outcome. Endosialin expression was highest in liposarcomas and lowest in leiomyosarcomas. A robust correlation between protein and gene expression data for both endosialin and PDGFR-ß was observed. Endosialin expression positively correlated with PDGFR-ß and heparin sulphate proteoglycan 2 and negatively correlated with carbonic anhydrase IX. Endosialin likely interacts with a network of extracellular and hypoxia activated proteins in sarcomas and other tumor types. Since expression does vary across histologic groups, endosialin may represent a selective target in soft tissue sarcomas.

Development of 89Zr-Ontuxizumab for in vivo TEM-1/endosialin PET applications.

PURPOSE: The complexity of sarcoma has led to the need for patient selection via in vivo biomarkers. Tumor endothelial marker-1 (TEM-1) is a cell surface marker expressed by the tumor microenvironment. Currently MORAb-004 (Ontuxizumab), an anti-TEM-1 humanized monoclonal antibody, is in sarcoma clinical trials. Development of positron emission tomography (PET) for in vivo TEM-1 expression may allow for stratification of patients, potentially enhancing clinical outcomes seen with Ontuxizumab. RESULTS: Characterization of cell lines revealed clear differences in TEM-1 expression. One high expressing (RD-ES) and one low expressing (LUPI) cell line were xenografted, and mice were injected with 89Zr-Ontuxizumab. PET imaging post-injection revealed that TEM-1 was highly expressed and readily detectable in vivo only in RD-ES. In vivo biodistribution studies confirmed high radiopharmaceutical uptake in tumor relative to normal organs. EXPERIMENTAL DESIGN: Sarcoma cell lines were characterized for TEM-1 expression. Ontuxizumab was labeled with 89Zr and evaluated for immunoreactivity preservation. 89Zr-Ontuxizumab was injected into mice with high or null expressing TEM-1 xenografts. In vivo PET imaging experiments were performed. CONCLUSION: 89Zr-Ontuxizumab can be used in vivo to determine high versus low TEM-1 expression. Reliable PET imaging of TEM-1 in sarcoma patients may allow for identification of patients that will attain the greatest benefit from anti-TEM-1 therapy.

Isolation of Circulating Tumor Cells from Multiple Epithelial Cancers with ApoStream(®) for Detecting (or Monitoring) the Expression of Folate Receptor Alpha.

This study describes our efforts to further the field of noninvasive diagnostics, specifically in the area of liquid biopsies in oncology. We employed laser scanning cytometry using highly selective antibodies to interrogate circulating tumor cells (CTCs) that were isolated using ApoStream(®) technology to identify folate receptor alpha (FRα)-positive cells. We demonstrate that FRα(+) CTCs can be isolated from patients with metastatic cancers, including NSCLC adenocarcinoma, breast cancer, and ovarian cancer, whereas squamous cell lung cancer and normal healthy controls were devoid of FRα(+) CTCs. We believe that the developed methodology will have applications in both the diagnosis and the monitoring of FRα-expressing cancers. Folate receptor alpha (FRα) expression may have utility as a potential diagnostic and therapeutic target in solid tumors. As tissue samples are not always available for patient screening, this study evaluated a noninvasive assay in CTCs from blood samples to detect FRα expression. The presence of FRα(+) CTCs enriched using ApoStream(®) and detected using laser capture cytometry was evaluated in blood samples from cancer patients [NSCLC adenocarcinoma (n = 14), breast cancer (n = 20), ovarian cancer (n = 6), and squamous lung cancer patients (n = 6)] and healthy subjects (n = 20). The data demonstrated that FRα(+) CTCs were detected in blood from NSCLC adenocarcinoma, breast, and ovarian cancer patients, whereas squamous cell lung cancer patients and normal healthy controls lacked FRα(+) CTCs as previously known. We demonstrate that CTCs captured using ApoStream(®) can be used to detect FRα(+) CTCs and may have clinical utility as a real-time liquid biopsy for assessing FRα levels in cancer patients.

Serum folate receptor alpha as a biomarker for ovarian cancer: Implications for diagnosis, prognosis and predicting its local tumor expression.

Folate receptor alpha (FRA) is a GPI-anchored glycoprotein and encoded by the FOLR1 gene. High expression of FRA is observed in specific malignant tumors of epithelial origin, including ovarian cancer, but exhibits very limited normal tissue expression, making it as an attractive target for the ovarian cancer therapy. FRA is known to shed from the cell surface into the circulation which allows for its measurement in the serum of patients. Recently, methods to detect the soluble form of FRA have been developed and serum FRA (sFRA) is considered a highly promising biomarker for ovarian cancer. We prospectively investigated the levels of sFRA in patients clinically suspected of having malignant ovarian tumors. A total of 231 patients were enrolled in this study and analyzed for sFRA as well as tumor expression of FRA by immunohistochemistry. High sFRA was predominantly observed in epithelial ovarian cancer patients, but not in patients with benign or borderline gynecological disease or metastatic ovarian tumors from advanced colorectal cancers. Levels of sFRA were highly correlated to clinical stage, tumor grade and histological type and demonstrated superior accuracy for the detection of ovarian cancer than did serum CA125. High sFRA was significantly associated with shorter progression-free survival in both early and advanced ovarian cancer patients. Finally, tumor FRA expression status was strongly correlated with sFRA levels. Taken together, these data suggest that sFRA might be a useful noninvasive serum biomarkers for future clinical trials assessing FRA-targeted therapy.

Endosialin Expression in Metastatic Melanoma Tumor Microenvironment Vasculature: Potential Therapeutic Implications.

Ontuxizumab (MORAb-004) is a humanized recombinant antibody targeting endosialin (TEM-1, CD248). We conducted an analysis of endosialin expression in metastatic melanoma specimens using the anti-endosialin rat anti- MAb 9G5, in order to determine the potential of endosialin as a therapeutic target within the tumor microenvironment vasculature. Endosialin expression in paraffin-embedded archival tissue block (PEAT) melanoma tissues was assessed using immunohistochemistry (IHC) with the anti-endosialin, MAb 9G5, in the vessels of American Joint Commission on Cancer (AJCC) Stage III (n = 18) and Stage IV (n = 48) specimens. IHC for endosialin expression was further performed on a TMA that included 136 Stage IV and 33 paired Stage III melanoma specimens. BRAF mutation (mt) was also evaluated in individual melanoma specimens and as well as the TMA. Analysis showed 70 % of melanoma specimens (n = 46) were positive for endosialin expression. There was no significant difference in endosialin and BRAFmt expression between stages III vs. IV specimens. Endosialin expression was detected in 86 % (n = 117) of stage IV TMA specimens, while no expression was detected in 29 normal tissue controls. MAb 9G5 detects the presence of endosialin in the microenvironment tumor vasculature of most metastatic melanoma tissues, regardless of clinical stage and presence of BRAFmt. Endosialin may be a potential therapeutic target by virtue of its selective expression in metastatic melanoma relative to normal tissues.

Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers.

BACKGROUND: Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues. METHODS: RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC. RESULTS: We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells. CONCLUSIONS: These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.

Folate receptor-α (FOLR1) expression and function in triple negative tumors.

Folate receptor alpha (FOLR1) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense overexpression of FOLR1 in breast tumors and poor prognosis, yet there is limited examination of the distribution of FOLR1 across clinically relevant breast cancer subtypes. To explore this further, we used RNA-seq data from multiple patient cohorts to analyze the distribution of FOLR1 mRNA across breast cancer subtypes comprised of estrogen receptor positive (ER+), human epidermal growth factor receptor positive (HER2+), and triple negative (TNBC) tumors. FOLR1 expression varied within breast tumor subtypes; triple negative/basal tumors were significantly associated with increased expression of FOLR1 mRNA, compared to ER+ and HER2+ tumors. However, subsets of high level FOLR1 expressing tumors were observed in all clinical subtypes. These observations were supported by immunohistochemical analysis of tissue microarrays, with the largest number of 3+ positive tumors and highest H-scores of any subtype represented by triple negatives, and lowest by ER+ tumors. FOLR1 expression did not correlate to common clinicopathological parameters such as tumor stage and nodal status. To delineate the importance of FOLR1 overexpression in triple negative cancers, RNA-interference was used to deplete FOLR1 in overexpressing triple negative cell breast lines. Loss of FOLR1 resulted in growth inhibition, whereas FOLR1 overexpression promoted folate uptake and growth advantage in low folate conditions. Taken together, our data suggests patients with triple negative cancers expressing high FOLR1 expression represent an important population of patients that may benefit from targeted anti-FOLR1 therapy. This may prove particularly helpful for a large number of patients who would typically be classified as triple negative and who to this point have been left without any targeted treatment options.

A first-in-human phase I study of MORAb-004, a monoclonal antibody to endosialin in patients with advanced solid tumors.

PURPOSE: Endosialin (TEM-1, CD248) is a protein expressed on the surface of activated mesenchymal cells, including certain subsets of tumors. Preclinical models suppressing endosialin function have shown antitumor activity. A humanized monoclonal antibody, MORAb-004, was engineered to target endosialin and is the first agent in clinical development for this mesenchymal cell target. EXPERIMENTAL DESIGN: This first-in-human, open-label, phase I study recruited patients with treatment-refractory solid tumors. MORAb-004 was administered intravenously once weekly in 4-week cycles. Objectives included determination of the safety of multiple infusions of MORAb-004, identification of the maximum tolerated dose (MTD), pharmacokinetic modeling, detection of any anti-human antibody response, and assessment of objective radiographic response to therapy. RESULTS: Thirty-six patients were treated at 10 dose levels of MORAb-004, ranging from 0.0625 to 16 mg/kg. Drug-related adverse events were primarily grade 1-2 infusion toxicities. Dose-limiting toxicity of grade 3 vomiting was observed at 16 mg/kg. Eighteen of 32 evaluable patients across all doses achieved disease stability, with minor radiographic responses observed in 4 patients (pancreatic neuroendocrine, hepatocellular, and sarcoma tumor types). Pharmacokinetics showed MORAb-004 accumulation beginning at 4 mg/kg and saturable elimination beginning at 0.25 mg/kg. Exposure increased in a greater-than-dose-proportional manner with terminal half-life increasing proportionally with dose. The MTD was identified as 12 mg/kg. CONCLUSIONS: Preliminary antitumor activity was observed. Safety profile, pharmacokinetics, and early antitumor activity suggest that MORAb-004 is safe at doses up to 12 mg/kg and should be studied further for efficacy.

Phase II clinical trial of amatuximab, a chimeric antimesothelin antibody with pemetrexed and cisplatin in advanced unresectable pleural mesothelioma.

PURPOSE: Amatuximab is a chimeric monoclonal antibody to mesothelin, a cell surface glycoprotein highly expressed in malignant pleural mesothelioma (MPM). On the basis of its synergy with chemotherapy in preclinical studies, we evaluated the antitumor activity of amatuximab plus pemetrexed and cisplatin in patients with unresectable MPM. EXPERIMENTAL DESIGN: In a single-arm phase II study, amatuximab (5 mg/kg) was administered on days 1 and 8 with pemetrexed (500 mg/m(2)) and cisplatin (75 mg/m(2)) on day 1 of 21-day cycles for up to six cycles. Patients with response or stable disease received amatuximab maintenance until disease progression. Primary endpoint was progression-free survival (PFS) at 6 months. Secondary endpoints were overall survival (OS), response rate, and safety. RESULTS: Eighty-nine patients were enrolled at 26 centers. Median of five cycles (range, 1-6) of combination treatment was administered, and 56 (63%) patients received amatuximab maintenance. Combination therapy resulted in no overlapping toxicities. Eleven patients (12.4%) had amatuximab-related hypersensitivity reactions. Responses included partial responses in 33 (40%) and stable disease in 42 (51%). Six-month PFS rate was 51% [95% confidence interval (CI), 39.1-62.3)], median PFS was 6.1 months (95% CI, 5.8-6.4), and median OS was 14.8 months (95% CI, 12.4-18.5) with 29 patients alive at data cut-off. CONCLUSIONS: Amatuximab with pemetrexed and cisplatin was well tolerated with objective tumor response or stable disease rate of 90% by independent radiologic review. Although PFS was not significantly different from historical controls, the median OS was 14.8 months with a third of patients alive and 5 continuing to receive amatuximab at the time of analysis.

Assessment of folate receptor-α and epidermal growth factor receptor expression in pemetrexed-treated non-small-cell lung cancer patients.

INTRODUCTION: Folate receptor-α regulates cellular uptake of folates and antifolates (eg, pemetrexed) and is frequently expressed in pulmonary adenocarcinoma. EGFR is an established therapeutic target in NSCLC. Therapies targeting FRA or EGFR are available. The association between FRA and EGFR expression in advanced NSCLC has not been explored. Combining therapeutic FRA antibodies with an EGFR inhibitor might be beneficial, if both of the targets are significantly coexpressed. PATIENTS AND METHODS: Specimens from 160 advanced NSCLC patients receiving pemetrexed-based chemotherapy were assessed for membranous FRA and EGFR protein expression using immunohistochemistry and the Hybrid (H)-score. EGFR (exons 18-21) and Kirsten RNA-associated rat sarcoma 2 virus (exon 2) mutations were determined. Results were correlated to patients' clinicopathological data, progression-free survival (PFS), and overall survival (OS). RESULTS: Forty-seven patients (29%) had tumors with strong FRA and EGFR expression, but no statistically significant correlation was seen between protein levels of FRA and EGFR. High membranous FRA expression (H-score ≥ 20) was associated with prolonged PFS (5.5 vs. 3.4 months; hazard ratio [HR], 0.6060; P = .0254) and improved OS (12.1 vs. 6.4 months; HR, 0.5726; P = .0076). CONCLUSION: Survival times are improved in NSCLC patients whose tumors show strong membranous FRA expression. No statistical correlation between membranous FRA and EGFR expression was demonstrated in advanced NSCLC, but 47 patients (29%) had higher expression of both of the receptors and could be suitable for combined targeted therapies.

Co-development of diagnostic vectors to support targeted therapies and theranostics: essential tools in personalized cancer therapy.

Novel technologies are being developed to improve patient therapy through the identification of targets and surrogate molecular signatures that can help direct appropriate treatment regimens for efficacy and drug safety. This is particularly the case in oncology whereby patient tumor and biofluids are routinely isolated and analyzed for genetic, immunohistochemical, and/or soluble markers to determine if a predictive biomarker signature (i.e., mutated gene product, differentially expressed protein, altered cell surface antigen, etc.) exists as a means for selecting optimal treatment. These biomarkers may be drug-specific targets and/or differentially expressed nucleic acids, proteins, or cell lineage profiles that can directly affect the patient's disease tissue or immune response to a therapeutic regimen. Improvements in diagnostics that can prescreen predictive response biomarker profiles will continue to optimize the ability to enhance patient therapy via molecularly defined disease-specific treatment. Conversely, patients lacking predictive response biomarkers will no longer needlessly be exposed to drugs that are unlikely to provide clinical benefit, thereby enabling patients to pursue other therapeutic options and lowering overall healthcare costs by avoiding futile treatment. While patient molecular profiling offers a powerful tool to direct treatment options, the difficulty in identifying disease-specific targets or predictive biomarker signatures that stratify a significant fraction within a disease indication remains challenging. A goal for drug developers is to identify and implement new strategies that can rapidly enable the development of beneficial disease-specific therapies for broad patient-specific targeting without the need of tedious predictive biomarker discovery and validation efforts, currently a bottleneck for development timelines. Successful strategies may gain an advantage by employing repurposed, less-expensive existing agents while potentially improving the therapeutic activity of novel, target-specific therapies that may otherwise have off-target toxicities or less efficacy in cells exhibiting certain pathways. Here, we discuss the use of co-developing diagnostic-targeting vectors to identify patients whose malignant tissue can specifically uptake a targeted anti-cancer drug vector prior to treatment. Using this system, a patient can be predetermined in real-time as to whether or not their tumor(s) can specifically uptake a drug-linked diagnostic vector, thus inferring the uptake of a similar vector linked to an anti-cancer agent. If tumor-specific uptake is observed, then the patient may be suitable for drug-linked vector therapy and have a higher likelihood of clinical benefit while patients with no tumor uptake should consider other therapeutic options. This approach offers complementary opportunities to rapidly develop broad tumor-specific agents for use in personalized medicine.