A new method for high speed, sensitive detection of minimal residual disease.

Investigations of rare cell types in peripheral blood samples, such as tumor, fetal, and endothelial cells, represent an emerging field with several potentially valuable medical applications. Peripheral blood is a particularly attractive body fluid for the detection of rare cells as its collection is minimally invasive and can be repeated throughout the course of the disease. Because the number of rare cells in mononuclear cells can be very low (1 in 10 million), a large number of cells must be quickly screened, which places demanding requirements on the screening technology. While enrichment technology has shown promise in managing metastatic disease, enrichment can cause distortions of cell morphology that limit pathological identification, and the enrichment targeting adds additional constraints that can affect sensitivity. Here, we describe a new approach for detecting rare leukemia cells that does not require prior enrichment. We have developed an immunocytochemical assay for identification of leukemia cells spiked in peripheral blood samples, and a high-speed scanning instrument with high numerical aperture and wide field of view to efficiently locate these cells in large sample sizes. A multiplex immunoassay with four biomarkers was used to uniquely identify the rare cells from leukocytes and labeling artifacts. The cytometer preserves the cell morphology and accurately locates labeled rare cells for subsequent high resolution imaging. The sensitivity and specificity of the approach show promise for detection of a low number of leukemia cells in blood (1 in 10 million nucleated cells). The method enables rapid location of rare circulating cells (25 M cells/min), no specific enrichment step, and excellent imaging of cellular morphology with multiple immunofluorescent markers. The cell imaging is comparable to other imaging approaches such as laser scan cytometry and image flow cytometry, but the cell analysis rate is many orders of magnitude faster making this approach practical for detection of rare cells.

Multiple biomarker expression on circulating tumor cells in comparison to tumor tissues from primary and metastatic sites in patients with locally advanced/inflammatory, and stage IV breast cancer, using a novel detection technology.

Patients with locally advanced/inflammatory breast cancer (LABC/IBC) face a high likelyhood of recurrence and prognosis for relapsed, or de novo stage IV metastatic breast cancer (MBC) remains poor. Estrogen (ER) and HER2 receptor expression on primary or MBC allow targeted therapies, but an estimated 10-18% of tumors do not exhibit these biomarkers and survival in these cases is even poorer. Variations in discordance rates for the expression of ER and HER2 receptors have been observed between primary and metastatic tumors and such discordances may lead to suboptimal treatment. Circulating tumor cells (CTCs) are considered the seeds of residual disease and distant metastases and their characterization could help guide treatment selection. To explore this possibility, we used multiple biomarker assessment of CTCs in comparison to primary and metastatic tumor sites. Thirty-six patients with LABC/IBC, or stage IV MBC were evaluated. Blood samples were procured prior to initiating or changing therapy. CTCs were identified based on presence of cytokeratin and nucleus staining, and the absence of CD45. A multimarker assay was developed to simultaneously quantify expression of HER2, ER, and ERCC1, a DNA excision repair protein. Novel fiber-optic array scanning technology (FAST) was used for sensitive location of CTCs. CTCs were detected in 82% of MBC and 62% LABC/IBC cases. Multiplex marker expression was successfully carried out in samples from18 patients with MBC and in 8 patients with LABC/IBC that contained CTCs. In MBC, we detected actionable discordance rates of 40 and 23%, respectively for ER and HER2 where a biomarker was negative in the primary or metastatic tumor and positive in the CTCs. In LABC/IBC, actionable discordances were 60 and 20% for ER and HER2, respectively. Pilot trials evaluating the effectiveness of treatment selections based on actionable discordances between biomarker expression patterns on CTCs and primary or metastatic tumor sites may allow for a prospective assessment of CTC-based individualized targeted therapies.

Measurement of enzyme kinetics and inhibitor constants using enthalpy arrays.

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. Compared with conventional calorimetry, enthalpy arrays achieve a significant reduction of sample volume and measurement time through the combination of the small size of the detectors and ability to perform measurements in parallel. The current capabilities of the technology for studying enzyme-catalyzed reactions are demonstrated by determining the kinetic parameters for reactions with three model enzymes. In addition, the technology has been used with two classes of enzymes to determine accurate inhibitor constants for competitive inhibitors from measurements at a single inhibitor concentration.

Glucose concentration monitoring using a small Fabry-Perot etalon.

Accurate measurements of aqueous glucose concentrations have been made in a double-chamber Fabry-Perot etalon that can be miniaturized for subcutaneous implantation to determine the concentration of glucose in interstitial fluid. In general, optical approaches to glucose detection measure light intensity, which in tissue varies due to inherent scattering and absorption. In our measurements, we compare the spectral positions of transmission maximums in two adjunct sections of an etalon in order to determine the refractive index difference between these sections and therefore we can tolerate large changes in intensity. With this approach, we were able to determine aqueous glucose concentrations between 0 mg/dl and 700 mg/dl within the precision of our reference measurement (+/-2.5 mg/dl or 2% of the measurement value). The use of reference cavities eliminates interference due to temperature variations, and we show the temperature independence over a temperature range of 32 degrees C to 42 degrees C. Furthermore, external filters eliminate interference from large molecule contaminants.

Enthalpy array analysis of enzymatic and binding reactions.

Enthalpy arrays enable label-free, solution-based calorimetric detection of molecular interactions in a 96-detector array format. The combination of the small size of the detectors and the ability to perform measurements in parallel results in a significant reduction of sample volume and measurement time compared with conventional calorimetry. We have made significant improvements in the technology by reducing the temperature noise of the detectors and improving the fabrication materials and methods. In combination with an automated measurement system, the advances in device performance and data analysis have allowed us to develop basic enzyme assays for substrate specificity and inhibitor activity. We have also performed a full titration of 18-crown-6 with barium chloride. These results point to future applications for enthalpy array technology, including fragment-based screening, secondary assays, and thermodynamic characterization of leads in drug discovery.

Structure and thermodynamic characterization of the EphB4/Ephrin-B2 antagonist peptide complex reveals the determinants for receptor specificity.

The Eph receptor tyrosine kinases and their ligands, the ephrins, regulate numerous biological processes in developing and adult tissues and have been implicated in cancer progression and in pathological forms of angiogenesis. We report the crystal structure of the EphB4 receptor in complex with a highly specific antagonistic peptide at a resolution of 1.65 angstroms. The peptide is situated in a hydrophobic cleft of EphB4 corresponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop, consistent with its antagonistic properties. Structural analysis identifies several residues within the EphB4 binding cleft that likely determine the ligand specificity of this receptor, while isothermal titration calorimetry experiments with truncated forms of the peptide define the amino acid residues of the peptide that are critical for receptor binding. These studies reveal structural features that will aid drug discovery initiatives to develop EphB4 antagonists for therapeutic applications.

Case study of the morphologic variation of circulating tumor cells.

We report a detailed cytomorphologic evaluation of the circulating component of widely metastatic breast carcinoma. A previously healthy 38-year-old woman was diagnosed with breast cancer. Wide local excision revealed a 1.7-cm infiltrating ductal adenocarcinoma, BSR score 7/9 with angiolymphatic invasion, and 4/20 lymph nodes positive for carcinoma. Five years later, a bone marrow biopsy revealed involvement of bone marrow by metastatic breast carcinoma, and shortly thereafter, metastases were identified in the liver and lung hilum. She enrolled in a clinical investigation for the detection of circulating tumor cells (CTCs) in breast carcinoma. A total of 659 CTCs were identified in a 10-mL blood sample using an immunofluorescent protocol targeting cytokeratins and detected using fiber-optic array scanning technology. The detected CTCs were subsequently stained with a Wright-Giemsa stain, and representative cells were evaluated in detail by light microscopy for morphologic evaluation. We find that the patient's CTCs exhibit a high degree of pleomorphism including CTCs with high and low nuclear-to-cytoplasmic ratios along with CTCs exhibiting early and late apoptotic changes. In addition, in comparison with her tumor cells in other sites, the full morphologic spectrum of cancer cells present in primary and metastatic tumor is also present in peripheral blood circulation.

High speed detection of circulating tumor cells.

Epithelial tumor cells circulate in peripheral blood at ultra-low concentrations in cancer patients. We have developed an instrument capable of rapid and accurate detection of rare cells in circulation utilizing fiber-optic array scanning technology (FAST). The FAST cytometer can locate immunofluorescently labeled rare cells on glass substrates at scan rates 500 times faster than conventional automated digital microscopy. These high scan rates are achieved by collecting fluorescent emissions using a fiber bundle with a large (50 mm) field of view. Very high scan rates make possible the ability to detect rare events without the requirement for an enrichment step. The FAST cytometer was used to detect, image and re-image circulating tumor cells in peripheral blood of breast cancer patients. This technology has the potential to serve as a clinically useful point-of-care diagnostic and a prognostic tool for cancer clinicians. The use of a fixed substrate permits the re-identification and re-staining of cells allowing for additional morphologic and biologic information to be obtained from previously collected and identified cells.

ERCC1 expression in circulating tumor cells (CTCs) using a novel detection platform correlates with progression-free survival (PFS) in patients with metastatic non-small-cell lung cancer (NSCLC) receiving platinum chemotherapy.

PURPOSE: To utilize a novel circulating tumor cell (CTC) technology to quantify ERCC1 expression on CTCs and determine whether ERCC1 expression levels predict efficacy of platinum-based chemotherapy in patients with metastatic non-small-cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: ERCC1 expression was measured in 17 metastatic NSCLC patients who received platinum-based therapy and had ≥2 intact CTCs with acceptable ERCC1 expression assay results. ERCC1 levels were determined from average expression on individual CTCs in each sample. Progression-free survival (PFS) was calculated from the date of therapy initiation. RESULTS: PFS decreased with increasing ERCC1 expression (p<0.04, F-test, linear regression). Lack of ERCC1 expression was associated with longer PFS (266 days versus 172 days, log-rank, p<0.02) in a Kaplan-Meier analysis using ERCC expression level of 1 as a cutoff (range 0-30). The difference in survival was statistically significant with a hazard ratio of 4.20 (95% CI 1.25-14.1, p<0.02, log-rank). PFS was also observed to decrease with increased cytokeratin (CK) expression (p<0.01 long-rank (Cox regression) and F-test (linear regression)). The hazard ratio is 4.38 (95% CI 1.76-10.9) for each log-change in CK value until progression was noted on imaging. CONCLUSION: Low expression of ERCC1 on CTCs correlates with PFS in patients with metastatic NSCLC receiving platinum-based therapy.

Rapid mixing of sub-microlitre drops by magnetic micro-stirring.

We demonstrate rapid mixing of sub-microlitre droplets (250 nl) using miniaturized magnetic stir bars (400 µm × 200 µm × 15 µm). The stir bars are fabricated using laser micromachining and placed on the substrate on which the drops are manipulated. They are activated by an externally applied magnetic field and used in combination with on-demand drop merging in enthalpy arrays. This technique results in a 10-fold increase in mixing rate, and a mixing time constant of about 2 s. Drop mixing times are measured by Förster resonance energy transfer (FRET) and verified by thermodynamic measurements of binding and enzymatic reactions.

Higher throughput calorimetry: opportunities, approaches and challenges.

Higher throughput thermodynamic measurements can provide value in structure-based drug discovery during fragment screening, hit validation, and lead optimization. Enthalpy can be used to detect and characterize ligand binding, and changes that affect the interaction of protein and ligand can sometimes be detected more readily from changes in the enthalpy of binding than from the corresponding free-energy changes or from protein-ligand structures. Newer, higher throughput calorimeters are being incorporated into the drug discovery process. Improvements in titration calorimeters come from extensions of a mature technology and face limitations in scaling. Conversely, array calorimetry, an emerging technology, shows promise for substantial improvements in throughput and material utilization, but improved sensitivity is needed.

Circulating tumor cells from well-differentiated lung adenocarcinoma retain cytomorphologic features of primary tumor type.

The detailed cytomorphologic appearance of circulating tumor cells (CTCs) in cancer patients is not well described, despite publication of multiple methods for enumerating these cells. In this case study, we present the cytomorphology of CTCs obtained from the blood of a woman with stage IIIB well-differentiated lung adenocarcinoma. Four years after she was diagnosed with her disease, 67 CTCs were identified in a blood sample using an immunofluorescent staining protocol and then subsequently stained with Wright-Giemsa. The cytomorphology of the CTCs was compared with the original tissue biopsy from 4 years prior. We found that CTCs and cells from the original biopsy had strikingly similar morphologic features, including large size in comparison to white blood cells and low nuclear to cytoplasmic ratios with voluminous cytoplasm. Careful cytomorphologic evaluation of CTCs will provide insights about the metastatic significance of these cells, which could yield widespread implications for the diagnosis, treatment, and management of cancer.

A rare-cell detector for cancer.

Although a reliable method for detection of cancer cells in blood would be an important tool for diagnosis and monitoring of solid tumors in early stages, current technologies cannot reliably detect the extremely low concentrations of these rare cells. The preferred method of detection, automated digital microscopy (ADM), is too slow to scan the large substrate areas. Here we report an approach that uses fiber-optic array scanning technology (FAST), which applies laser-printing techniques to the rare-cell detection problem. With FAST cytometry, laser-printing optics are used to excite 300,000 cells per sec, and emission is collected in an extremely wide field of view, enabling a 500-fold speed-up over ADM with comparable sensitivity and superior specificity. The combination of FAST enrichment and ADM imaging has the performance required for reliable detection of early-stage cancer in blood.

Enthalpy arrays.

We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein-ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein-ligand binding interactions (RNase A + cytidine 2'-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler.