Benjamin A. Katchman

Analysis of the Plasma Peptidome from Pancreas Cancer Patients Connects a Peptide in Plasma to Overexpression of the Parent Protein in Tumors

Blood circulates through nearly every organ including tumors. Therefore, plasma is a logical source to search for tumor-derived proteins and peptides. The challenge with plasma is that it is a complex bodily fluid composed of high concentrations of normal host proteins that obscure identification of tumor-derived molecules. To simplify plasma, we examined a low molecular weight (LMW) fraction (plasma peptidome) using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. In the plasma peptidome of patients with ductal adenocarcinoma of the pancreas (DAP), a prominent peptide was identified from the QSOX1 parent protein. This peptide is stable in whole blood over 24 h and was present in 16 of 23 DAP patients and 4 of 5 patients with intraductal papillary mucinous neoplasm (IPMN). QSOX1 peptides were never identified in the plasma peptidome from 42 normal healthy donors using the same methods. Immunohistochemical staining of DAP tissue sections with anti-QSOX1 antibody shows overexpression of QSOX1 in tumor but not in adjacent stroma or normal ducts. Three of four pancreas tumor cell lines also express QSOX1 protein by Western blot analysis. This is the first report of QSOX1 peptides in plasma from DAP patients and makes the rare connection between a peptide in plasma from cancer patients and overexpression of the parent protein in tumors.

Quiescin Sulfhydryl Oxidase 1 Promotes Invasion of Pancreatic Tumor Cells Mediated by Matrix Metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9. Mol Cancer Res; 9(12); 1621–31. ©2011 AACR.

Application of Flexible OLED Display Technology to Point-of-Care Medical Diagnostic Testing

This paper presents a new concept combining flexible organic light-emitting diode (OLED) display technology with fluorescent biorecognition microarray technology to fabricate point-of-care immunobiosensors. Our approach is designed to leverage commercial OLED display technology to reduce pre-functionalized biosensor substrate costs to pennies per cm2 combined with leveraging the display industries ability to manufacture an immense number of low-cost consumer electronic products annually. For this work, we demonstrate that our new approach using high brightness flexible OLED display technology combined with a charge integrating readout circuit and optical filters can offer point-of-care diagnostic sensitivity at or below 10 pg/mL, which approaches the lower limit of detection (LLOD) of typical clinical laboratory instrumentation.

Autoantibody biomarkers for the detection of serous ovarian cancer

Objective The purpose of this study was to identify a panel of novel serum tumor antigen-associated autoantibody (TAAb) biomarkers for the diagnosis of high-grade serous ovarian cancer. METHODS To detect TAAb we probed high-density programmable protein microarrays (NAPPA) containing 10,247 antigens with sera from patients with serous ovarian cancer (n=30 cases/30 healthy controls) and measured bound IgG. We identified 735 promising tumor antigens and evaluated these with an independent set of serous ovarian cancer sera (n=30 cases/30 benign disease controls/30 healthy controls). Thirty-nine potential tumor autoantigens were identified and evaluated using an orthogonal programmable ELISA platform against a total of 153 sera samples (n=63 cases/30 benign disease controls/60 healthy controls). Sensitivities at 95% specificity were calculated and a classifier for the detection of high-grade serous ovarian cancer was constructed. RESULTS We identified 11-TAAbs (ICAM3, CTAG2, p53, STYXL1, PVR, POMC, NUDT11, TRIM39, UHMK1, KSR1, and NXF3) that distinguished high-grade serous ovarian cancer cases from healthy controls with a combined 45% sensitivity at 98% specificity. CONCLUSION These are potential circulating biomarkers for the detection of serous ovarian cancer, and warrant confirmation in larger clinical cohorts.

Application of flat panel OLED display technology for the point-of-care detection of circulating cancer biomarkers

Point-of-care molecular diagnostics can provide efficient and cost-effective medical care, and they have the potential to fundamentally change our approach to global health. However, most existing approaches are not scalable to include multiple biomarkers. As a solution, we have combined commercial flat panel OLED display technology with protein microarray technology to enable high-density fluorescent, programmable, multiplexed biorecognition in a compact and disposable configuration with clinical-level sensitivity. Our approach leverages advances in commercial display technology to reduce pre-functionalized biosensor substrate costs to pennies per cm2. Here, we demonstrate quantitative detection of IgG antibodies to multiple viral antigens in patient serum samples with detection limits for human IgG in the 10 pg/mL range. We also demonstrate multiplexed detection of antibodies to the HPV16 proteins E2, E6, and E7, which are circulating biomarkers for cervical as well as head and neck cancers.

Proteomic mapping of p53 immunogenicity in pancreatic, ovarian, and breast cancers.

Mutations in TP53 induce autoantibody immune responses in a subset of cancer patients, which have been proposed as biomarkers for early detection. Here, we investigate the association of p53‐specific autoantibodies with multiple tumor subtypes and determine the association with p53 mutation status and epitope specificity.

Disposable point-of-use optical biosensor for multiple biomarker detection

This work explored the viability of a new miniaturized fluorescence measurement-based sensor architecture using organic light emitting diode (OLED) display and photodiode active matrix array technology for point-of-use diagnosis of multiple disease biomarkers in a low cost disposable configuration. Sensor feasibility and optical performance were evaluated using a bright 0.3mW/mm2 2 × 2 mm, 455nm blue OLED emitter test structure configured first with orthogonally crossed linear polarizing film; and second, with band-pass and long-pass optical filters. Preliminary measurement results indicated that this type of sensor architecture requires optical filters to approach the sensitivity of laboratory fluorescence-based instrumentation.

Programmable protein arrays for immunoprofiling HPV-associated cancers.

Over 600 000 cancers each year are attributed to the human papillomavirus (HPV), including cervical, anogenital and oropharyngeal cancers (OPC). A key challenge in understanding HPV immunobiology is the diversity of oncogenic HPV types and the need for multiplexed display of HPV antigens to measure antibody responses. We have generated custom HPV protein microarrays displaying 98 proteins as C‐terminal GST fusion proteins, representing eight antigens of two low‐risk HPV types (HPV6 and 11) and ten oncogenic high‐risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52 and 58). We demonstrate robust and reproducible protein expression of 96/98 of the antigens using a human cell lysate expression system. The target epitopes and specificities of four monoclonal antibodies were identified. Using sera from ten patients with newly diagnosed OPC and ten controls, we demonstrate specific IgG seroreactivity to HPV16 E1, E2, and E7 (a fold increase of 1.52, 2.19 and 1.35 in cases vs. controls, respectively, all p < 0.005), confirming our prior data on an ELISA platform. We also detect HPV52 E7 Abs in serum from a patient with cervical cancer. The HPV protein array has potential for rapid identification of serologic responses to 12 HPV types.

Characterization of a compact and highly sensitive fluorescence-based detection system for point-of-care applications

This work details the design and characterization of a low-cost, portable and highly sensitive fluorescence detection system intended for use in a compact and disposable point-of-care (POC) device. The detection device leverages time integration to improve the signal to noise ratio (SNR) compared to instantaneous measurements. It also eliminates complicated focusing optics and electronics typically found in bulky and expensive laboratory-scale devices. Characterization was performed by measuring a series of logarithmically scaled dilutions of 1 μm Nile Red fluorescent microspheres immobilized on microscope slides. This approach eliminates assay dependencies and elucidates the actual system performance. A theoretical model that predicts the time-integrated output voltage profile of the sensor was developed; this prediction is useful for evaluating any lens-free fluorescent system based on a set of filters and choice of fluorophore. By relating the fluorophore concentration, system design parameters, and the output voltage, the model matches well with the empirical data and the limit of determination (LOD) for Nile Red is 20 particles. This system provides a sensitive and potentially low-cost device for fluorescent diagnosis in an integrated and compact/miniaturized POC device, lab-on-chip or a table-top reader.

Low-cost, disposable fluorescence-based biorecognition system architecture for multiplexed point-of-care molecular diagnostics

The high per patient cost of quantitative, high-sensitivity molecular diagnostics is one of the key roadblocks limiting the transition of this technology from the clinical laboratory to point-of-care diagnostics in low-to-middle income countries (LMICs). As a solution, we present a low-cost system architecture for fluorescence-based point-of-care diagnostics using small volume patient sera samples. Our approach combines an inexpensive 4-site microscope slide reader with low per patient cost consumables using conventional glass microscope slides, pre-printed with biorecognition molecules. We use a neuromorphic-inspired, charge-integrating readout circuit architecture combined with optical filters in a sandwich-style configuration to achieve high-sensitivity while trading off detection time and number of biorecognition sites per slide. Our demonstrated lower limit of detection using Nile Red fluorescent microspheres was a 1,000,000:1 dilution corresponding to approximately 200 microspheres per detection site, approaching the sensitivity of a conventional high-cost desktop clinical laboratory microscope slide reader.