Standardization of flow cytometric detection of antigen expression.

Since response to antigen-based immunotherapy relies upon the level of tumor antigen expression we developed an antigen quantification assay using ABC values. Antigen quantification as a clinical assay requires methods for quality control and for interlaboratory and inter-cytometer platform standardization. A single lot of Cytotrol™ Lyophilized Control Cells (Beckman Coulter) used for all studies. The variability in antigen quantification across 4 different instrument platforms in 2 separate laboratories was evaluated. The effect of the antibody clone utilized, importance of custom 1:1 molar ratio (fluorophore to protein, F/P) verses off-the-shelf antibodies, and QuantiBrite PE calibration verses linearity calibration combined with a single point scale transformation with CD4 as reference were determined. Use of single lot control cells allowed validation of reproducibility between flow cytometer platforms and laboratories and allowed assessment of different antibody lots, cocktail preparation, and different antibody clones. Off the shelf antibody preparations provide reproducible estimates of antigen density, however custom 1:1 unimolar antibody preparations should be utilized for definitive measurement of antigen expression.Geometric Mean fluorescent Intensity (GeoMFI) was not comparable across instruments and inter-laboratory. The use of CD4 as the reference marker can minimize variability in ABC values. Comparable antigen quantification is vital in managing patients receiving antigen-based immunotherapy. If this assay is to be utilized in a clinical setting, quality control methods have to be instituted to assure reproducibility and allow validation across laboratories. We have demonstrated that use of a lyophilized cell control is highly valuable in achieveing these goals.

The Impact of Tumor Treating Fields on Glioblastoma Progression Patterns.

PURPOSE: Tumor-treating fields (TTFields) are an antimitotic treatment modality that interfere with glioblastoma (GBM) cell division and organelle assembly by delivering low-intensity, alternating electric fields to the tumor. A previous analysis from the pivotal EF-14 trial demonstrated a clear correlation between TTFields dose density at the tumor bed and survival in patients treated with TTFields. This study tests the hypothesis that the antimitotic effects of TTFields result in measurable changes in the location and patterns of progression of newly diagnosed GBM. METHODS AND MATERIALS: Magnetic resonance images of 428 newly diagnosed GBM patients who participated in the pivotal EF-14 trial were reviewed, and the rates at which distant progression occurred in the TTFields treatment and control arm were compared. Realistic head models of 252 TTFields-treated patients were created, and TTFields intensity distributions were calculated using a finite element method. The TTFields dose was calculated within regions of the tumor bed and normal brain, and its relationship with progression was determined. RESULTS: Distant progression was frequently observed in the TTFields-treated arm, and distant lesions in the TTFields-treated arm appeared at greater distances from the primary lesion than in the control arm. Distant progression correlated with improved clinical outcome in the TTFields patients, with no such correlation observed in the controls. Areas of normal brain that remained normal were exposed to higher TTFields doses compared with normal brain that subsequently exhibited neoplastic progression. Additionally, the average dose to areas of the enhancing tumor that returned to normal was significantly higher than in the areas of the normal brain that progressed to enhancing tumor. CONCLUSIONS: There was a direct correlation between TTFields dose distribution and tumor response, confirming the therapeutic activity of TTFields and the rationale for optimizing array placement to maximize the TTFields dose in areas at highest risk of progression, as well as array layout adaptation after progression.

Development and Validation of Measurement Traceability for In Situ Immunoassays.

BACKGROUND: Immunoassays for protein analytes measured in situ support a $2 billion laboratory testing industry that suffers from significant interlaboratory disparities, affecting patient treatment. The root cause is that immunohistochemical testing lacks the generally accepted tools for analytic standardization, including reference standards and traceable units of measure. Until now, the creation of these tools has represented an insoluble technical hurdle. METHODS: We address the need with a new concept in metrology-that is, linked traceability. Rather than calculating analyte concentration directly, which has proven too variable, we calculate concentration by measuring an attached fluorescein, traceable to NIST Standard Reference Material 1934, a fluorescein standard. RESULTS: For validation, newly developed estrogen receptor (ER) calibrators were deployed in tandem with an array of 80 breast cancer tissue sections in a national external quality assessment program. Laboratory performance was assessed using both the ER standards and the tissue array. Similar to previous studies, the tissue array revealed substantial discrepancies in ER test results among the participating laboratories. The new ER calibrators revealed a broad range of analytic sensitivity, with the lower limits of detection ranging from 7310 to 74 790 molecules of ER. The data demonstrate, for the first time, that the variable test results correlate with analytic sensitivity, which can now be measured quantitatively. CONCLUSIONS: The reference standard enables precise interlaboratory alignment of immunohistochemistry test sensitivity for measuring cellular proteins in situ. The introduction of a reference standard and traceable units of measure for protein expression marks an important milestone.

ACR Appropriateness Criteria® definitive external beam irradiation in stage T1 and T2 prostate cancer.

PURPOSE: : External beam radiation therapy is a standard of care treatment for men who present with clinically localized (T1-T2) prostate cancer. The purpose of this review was to provide clarification on the appropriateness criteria and management considerations for the treatment of prostate cancer with external beam radiation therapy. METHODS: : A panel consisting of physicians with expertise on prostate cancer was assembled and provided with a number of clinical scenarios for consensus treatment and management guidelines. Prostate cancer patient vignettes were presented along with specific management recommendations based on an extensive review of the modern external beam radiotherapy literature. The American College of Radiology Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer reviewed journals and the application of a well established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances, where evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment. RESULTS: : Modern external beam radiation therapy series demonstrate favorable biochemical control rates for patients with localized prostate cancer. Morbidity profiles are also favorable and it is clear that this is enhanced by modern techniques like 3-dimensional conformal radiation therapy and intensity-modulated radiation therapy. An active area of investigation is evaluating the use of hypofractionated dosing. CONCLUSIONS: : Continued investigation to refine patient selection, external beam radiation technology application, and alternative dosing schedules should result in further improvements in biochemical outcome and decreased morbidity with external beam radiation treatment for localized prostate cancer.

Development of bavituximab, a vascular targeting agent with immune-modulating properties, for lung cancer treatment.

Bavituximab is a chimeric monoclonal antibody directed against the membrane phospholipid phosphatidylserine. Phosphatidylserine exposure is increased on endothelial cells and apoptotic cancer cells in solid tumors, allowing tumor-specific targeting of bavituximab. Bavituximab binding results in tumor vessel occlusion and enhanced antitumor immunity. Preclinical investigations have demonstrated efficacy as monotherapy and in combination with other modalities against multiple cancer types. Phase I clinical trials of bavituximab monotherapy and in combination with chemotherapy in adults with refractory solid tumors have been completed. Phase II trials of bavituximab in combination with chemotherapy for the first- and second-line treatment of advanced non-small-cell lung cancer are currently ongoing. This article summarizes the preclinical development and clinical experience with bavituximab in non-small-cell lung cancer.

Phase I dose-escalation study of stereotactic body radiation therapy for low- and intermediate-risk prostate cancer.

PURPOSE: To evaluate the tolerability of escalating doses of stereotactic body radiation therapy in the treatment of localized prostate cancer. PATIENTS AND METHODS: Eligible patients included those with Gleason score 2 to 6 with prostate-specific antigen (PSA) ≤ 20, Gleason score 7 with PSA ≤ 15, ≤ T2b, prostate size ≤ 60 cm(3), and American Urological Association (AUA) score ≤ 15. Pretreatment preparation required an enema and placement of a rectal balloon. Dose-limiting toxicity (DLT) was defined as grade 3 or worse GI/genitourinary (GU) toxicity by Common Terminology Criteria of Adverse Events (version 3). Patients completed quality-of-life questionnaires at defined intervals. RESULTS: Groups of 15 patients received 45 Gy, 47.5 Gy, and 50 Gy in five fractions (45 total patients). The median follow-up is 30 months (range, 3 to 36 months), 18 months (range, 0 to 30 months), and 12 months (range, 3 to 18 months) for the 45 Gy, 47.5 Gy, and 50 Gy groups, respectively. For all patients, GI grade ≥ 2 and grade ≥ 3 toxicity occurred in 18% and 2%, respectively, and GU grade ≥ 2 and grade ≥ 3 toxicity occurred in 31% and 4%, respectively. Mean AUA scores increased significantly from baseline in the 47.5-Gy dose level (P = .002) as compared with the other dose levels, where mean values returned to baseline. Rectal quality-of-life scores (Expanded Prostate Cancer Index Composite) fell from baseline up to 12 months but trended back at 18 months. In all patients, PSA control is 100% by the nadir + 2 ng/mL failure definition. CONCLUSION: Dose escalation to 50 Gy has been completed without DLT. A multicenter phase II trial is underway treating patients to 50 Gy in five fractions to further evaluate this experimental therapy.

Microarray analyses during adipogenesis: understanding the effects of Wnt signaling on adipogenesis and the roles of liver X receptor alpha in adipocyte metabolism.

Wnt signaling maintains preadipocytes in an undifferentiated state. When Wnt signaling is enforced, 3T3-L1 preadipocytes no longer undergo adipocyte conversion in response to adipogenic medium. Here we used microarray analyses to identify subsets of genes whose expression is aberrant when differentiation is blocked through enforced Wnt signaling. Furthermore, we used the microarray data to identify potentially important adipocyte genes and chose one of these, the liver X receptor alpha (LXR alpha), for further analyses. Our studies indicate that enforced Wnt signaling blunts the changes in gene expression that correspond to mitotic clonal expansion, suggesting that Wnt signaling inhibits adipogenesis in part through dysregulation of the cell cycle. Experiments designed to uncover the potential role of LXR alpha in adipogenesis revealed that this transcription factor, unlike CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma, is not adipogenic but rather inhibits adipogenesis if inappropriately expressed and activated. However, LXR alpha has several important roles in adipocyte function. Our studies show that this nuclear receptor increases basal glucose uptake and glycogen synthesis in 3T3-L1 adipocytes. In addition, LXR alpha increases cholesterol synthesis and release of nonesterified fatty acids. Finally, treatment of mice with an LXR alpha agonist results in increased serum levels of glycerol and nonesterified fatty acids, consistent with increased lipolysis within adipose tissue. These findings demonstrate new metabolic roles for LXR alpha and increase our understanding of adipogenesis.