Donald H. Atha (Assoc)

Histopathology is the hallmark for diagnostic and therapeutic decisions in almost all forms of cancer (Collan 1984; Apple 2006). Histological methods are widely used because they provide in situ spatial and biochemical information. However, pathologists in routine practice may have problems recognizing some of the more subtle criteria and utilizing these data for diagnostic purposes. The manual interpretation of histological sections can be subjective; sometimes involving a wide spectrum of clinical interpretation where the final morphologic assessment lacks a definitive diagnosis (Taylor 2000). Some cancers are more difficult than others to interpret, resulting in a need for a quantitative basis to differentiate and bin types of cancer, stage of cancer progression, cellular phenotypes and atypical lesions (Tosi and Cottier 1989; Maruvada et al. 2005; O'Connell 2005). Standard methods and reference materials for immunohistological methods would be valuable tools for pathologists to subtype specific cancers and categorize some of the rarer tumors (Gil and Wu 2003). More objective approaches, using standard methods and reference materials, will reduce inherent subjectivity and facilitate the accurate interpretation of histological features. This may expedite the acceptance of certain biomarkers, such as telomerase, for diagnostic use (Wang et al. 2005).

Human telomerase reverse transcriptase (hTERT) is a multi-component enzyme that catalyzes the end capping of the chromosomes (telomeres) with the repetitive DNA sequence motif (TTAGGG) (Blackburn 1991; Levy et al. 1992). It maintains chromosomal length and stability by this process, thereby promoting cellular immortalization. It is expressed during development and in 85% to 90 % of all human cancers, but not in normal adult, non-stem cell somatic tissue, which makes it an attractive tumor diagnostic marker (see review, Shay 1999). However, proteomic and genomic assays for telomerase have been cumbersome and without a universally accepted standard to compare between laboratories. Previously, we have improved the sensitivity and reproducibility of the telomerase repeat amplification protocol (TRAP) assay system using capillary electrophoresis and a high-throughput RApidTRAP (robot-assisted TRAP) system (Atha et al. 2003; Jakupciak et al. 2004), and have reported a candidate telomerase reference material for TRAP and RT-PCR based assays (Jakupciak et al. 2005). This well-characterized candidate reference material was evaluated for potential diagnostic use across biochemical-based and direct imaging-based technologies for the purposes of developing immunohistological reference materials. The clinical applications of these reference materials were evaluated to define thresholds of telomerase activity present in esophageal balloon cytology species from a population at risk for esophageal cancer (McGruder et al. 2006).

Correlating proteomic and genomic assays to immunohistochemical (IHC) detection of telomerase, which has been widely employed on cancerous tissue sections, histoids, cell lines and tumor cells, albeit with mixed results, is a powerful approach to biomarker development (Elkak et al., 2005). The reliability of the available antibodies has been repeatedly brought into question limiting the acceptance of telomerase as a biomarker (Wu et al. 2006). Furthermore, the crystal structure of telomerase has not been elucidated and the fragments that have been used as the immunostimulant for the generation of antibodies have consisted of relatively short amino acid sequences at the C- or N- terminus. Therefore identifying the location, affinity and specificity of binding has proven difficult. To this end we have used an automated fluorescent microscopy method to measure the respective staining patterns and heterogeneities of three different commercially available antibodies. By employing automated methods we are minimizing user bias and collecting data on sufficient numbers of cells to discriminate easily between telomerase levels in cultured human lung tumor cells and in normal human fibroblast cells as a control. Although the measurement of telomerase by antibody staining does not determine whether the telomerase is active, it is a valuable tool to determine its presence in individual cells, where tissue heterogeneity can prevent accurate diagnosis.

To determine telomerase activity, we have compared our immunohistochemical measurements to TRAP/PCR using our RApidTRAP system and telomerase candidate reference materials. We also have compared the cancer and control cell lines used in our IHC measurements using RT-PCR to determine the levels of telomerase mRNA. This has ensured that the cancer and control cell lines used in our analysis contain the expected levels of telomerase. In addition, we have used flow cytometry to compare the anti-telomerase antibodies used in our analysis (Jakupciak et al. 2008). Such a combination of measurement methods has not only provided a more complete quantitation of telomerase but also is providing a model for evaluating other candidate biomarkers associated with cancer.

Selected Publications (Telomerase and p53):

Atha, D.H., Wenz, H-M., Morehead, H., Tian, J. and O'Connell, C. Detection of p53 Point Mutations by Single Strand Conformation Polymorphism (SSCP): Analysis by Capillary Electrophoresis. Electrophoresis, 19, 172 -179 (1998).

O'Connell, C.D., Tian, J., Juhasz, A., Wenz, H.-M. and Atha, D.H. Development of Standard Reference Materials for Diagnosis of P53 Mutations: Analysis by Slab Gel-SSCP. Electrophoresis, 19, 164 -171 (1998).

Atha, D.H. Characterization of DNA Standards by Capillary Electrophoresis.  Electrophoresis, 19, 1428-1435 (1998).

Wenz, H.-M, Ramachandra, S., O'Connell, C.D. and Atha, D.H. Identification of known p53 point mutations by capillary electrophoresis using unique mobility profiles in a blinded study, Mutation Research Genomics, 382, 1-132 (1998).

Atha, D.H. Kasprzak, W., O'Connell, C.D. and Shapiro, B.A. Prediction of DNA Single Strand Conformational Polymorphism (SSCP): Analysis by Capillary Electrophoresis and Computerized DNA Modeling, Frederick Conference on Capillary Electrophoresis, Nucleic Acids Research, 29, 4643-4653 (2001).

Atha, D.H., Miller, K., Sanow, A.D., Xu, J. Hess, J.L., Wu, O.C., Wang, W., Srivastava, S., Highsmith, W.E. High-Throughput Analysis of Telomerase by Capillary Electrophoresis, Electrophoresis, 24, 109-114 (2003).

O'Connell, C.D., Tully, L.A., Devaney, J.M., Marino, M.A., Jakupciak, J.P. and Atha, D.H. Renewable Standard Reference Material for the Detection of TP53 Mutations. Molecular Diagnosis, 7, 85-97 (2003).

Hess, J.L., Atha, D.H., Xu, J., and Highsmith, W.E. Telomerase Activity Measurement in Magnetically-Captured Epithelial Cells: Comparison of Slab-Gel and Capillary Electrophoresis, Electrophoresis, 25, 1852-1859 (2004).

Jakupciak, J.P., Wang, W., Barker, P.E. Srivastava, S. and Atha, D.H. Analytical Validation of Telomerase Activity for Cancer Early Detection: TRAP/PCR-CE and hTERT mRNA Quantification Assay for High-throughput Screening of Tumor Cells, J. Molecular Diagnostics, 6, 157-165 (2004).

Sunar-Reeder, B., Atha, D.H., Aydemir, S., Reeder, D.J., Tully, L., Khan, R. and O'Connell, C.D. Use of TP53 Reference Materials to Validate Mutations in Clinical Tissue Specimens by Single-Strand Conformational Polymorphism, Molecular Diagnosis, 8, 123-130 (2004).

Jakupciak, J.P., Barker, P.E., Wang, W., Srivastava, S. and Atha, D.H. Preparation and Characterization of Candidate Reference Materials for Telomerase Assays, Clinical Chemistry, 51, 1443-1450 (2005).

O'Connell, C.D., Atha, D.H., and Jakupciak, J.P. Standards for Validation of Cancer Biomarkers, Cancer Biomarkers, 1, 233-239 (2005).

McGruder, B.M., Atha, D.H., Wang, W., Huppi, K., Wei, W-Q., Abnet, C.C., Qiao, Y-L., Dawsey, S.M., Taylor, P.R., and Jakupciak, J.P. Real-time Telomerase Assay of Less-Invasive Collected Samples, Cancer Letters, 244, 91-100 (2006).

Reipa, V., Niaura, G., and Atha, D.H. Conformational Analysis of the Telomerase RNA Pseudoknot Hairpin by Raman Spectroscopy, RNA, 13, 108-115 (2007).

Atha, D.H. High-Throughput DNA Diagnostic Measurements using Capillary Electrophoresis: p53, Fragile X and Telomerase. Expert Opin. Med. Diagn. 2, 91-100 (2008).

Jakupciak, J.P., Gallant, N.D., Smith, A.H., Becker, M.L., Tona, A., and Atha, D.H. Improved Methods and Standards for Telomerase Detection: Quantitative Histopathology using Antibody Staining. Biotechnic and Histochemistry, 2008 in press