Jose I. Diaz

Lectin Capture Strategies Combined with Mass Spectrometry for the Discovery of Serum Glycoprotein Biomarkers

The application of mass spectrometry to identify disease biomarkers in clinical fluids like serum using high throughput protein expression profiling continues to evolve as technology development, clinical study design, and bioinformatics improve. Previous protein expression profiling studies have offered needed insight into issues of technical reproducibility, instrument calibration, sample preparation, study design, and supervised bioinformatic data analysis. In this overview, new strategies to increase the utility of protein expression profiling for clinical biomarker assay development are discussed with an emphasis on utilizing differential lectin-based glycoprotein capture and targeted immunoassays. The carbohydrate binding specificities of different lectins offer a biological affinity approach that complements existing mass spectrometer capabilities and retains automated throughput options. Specific examples using serum samples from prostate cancer and hepatocellular carcinoma subjects are provided along with suggested experimental strategies for integration of lectin-based methods into clinical fluid expression profiling strategies. Our example workflow incorporates the necessity of early validation in biomarker discovery using an immunoaffinity-based targeted analytical approach that integrates well with upstream discovery technologies.

Phase I trial of a B7-1 (CD80) gene modified autologous tumor cell vaccine in combination with systemic interleukin-2 in patients with metastatic renal cell carcinoma.

PURPOSE A reason that the immune system may fail to reject tumors is that T cells encounter tumor antigen derived peptides on the surface of tumor cells in a tolerizing rather than activating context since tumor cells do not express T cell co-stimulatory molecules such as B7-1 (CD80). In preclinical models over expression of B7-1 on the surface of tumor cells has been shown to activate T cells which kill tumor cells. We conducted a phase I clinical trial testing this approach in patients with metastatic renal cell carcinoma. MATERIALS AND METHODS Resected tumors from 15 patients were disaggregated and adapted to tissue culture, transduced with the B7-1 gene and injected subcutaneously as a vaccine. The dose of the vaccine was escalated in 3 separate cohorts of patients, and systemic interleukin-2 (IL-2) was administered as an adjuvant designed to enhance the proliferation of the vaccine activated T cells. RESULTS Of the 15 patients 9 had measurable disease, 2 had a partial response and 2 had stable disease. Perivascular T cell infiltrates at autologous tumor delayed type hypersensitivity skin test sites developed in 3 of the 4 patients with stable disease or partial response. Although the patients experienced the usual and expected toxicity from the IL-2, there was no significant toxicity observed with the vaccine. CONCLUSIONS The B7-1 gene modified autologous tumor cell vaccine is safe and can be combined with systemic IL-2 with acceptable toxicity. Immunological and clinical responses were observed in some of the patients. A phase II trial is reasonable to determine the efficacy of this approach.

Search for potential markers for prostate cancer diagnosis, prognosis and treatment in clinical tissue specimens using amine-specific isobaric tagging (iTRAQ) with two-dimensional liquid chromatography and tandem mass spectrometry

This study aimed to identify candidate new diagnosis and prognosis markers and medicinal targets of prostate cancer (PCa), using state of the art proteomics. A total of 20 prostate tissue specimens from 10 patients with benign prostatic hyperplasia (BPH) and 10 with PCa (Tumour Node Metastasis [TNM] stage T1-T3) were analyzed by isobaric stable isotope labeling (iTRAQ) and two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) approaches using a hybrid quadrupole time-of-flight system (QqTOF). The study resulted in the reproducible identification of 825 nonredundant gene products (p < or = 0.05) of which 30 exhibited up-regulation (> or =2-fold) and another 35 exhibited down-regulation (< or =0.5-fold) between the BPH and PCa specimens constituting a major contribution toward their global proteomic assessment. Selected findings were confirmed by immunohistochemical analysis of prostate tissue specimens. The proteins determined support existing knowledge and uncover novel and promising PCa biomarkers. The PCa proteome found can serve as a useful aid for the identification of improved diagnostic and prognostic markers and ultimately novel chemopreventive and therapeutic targets.

DNA Damage–Induced Protein 14-3-3 σ Inhibits Protein Kinase B/Akt Activation and Suppresses Akt-Activated Cancer

14-3-3 sigma is induced by tumor suppressor protein p53 in response to DNA damage. p53 can directly transactivate the expression of 14-3-3 sigma to cause a G(2) cell cycle arrest when cell DNA is damaged. The expression of 14-3-3 sigma protein is down-regulated in various tumors, but its function has not been fully established. Protein kinase B/Akt, a crucial regulator of oncogenic signal involved in cell survival and proliferation, is deregulated in many types of cancer. Akt activation can enhance p53 degradation, but its role in DNA damage response is not clear. Here, we show that Akt activation is diminished when p53 and 14-3-3 sigma is up-regulated in response to DNA damage. Evidence is provided that 14-3-3 sigma binds and inhibits Akt. In keeping with this concept, Akt-mediated cell survival is inhibited by 14-3-3 sigma. Significantly, we show that 14-3-3 sigma inhibits Akt-mediated cell growth, transformation, and tumorigenesis. Low expression of 14-3-3 sigma in human primary breast cancers correlates with Akt activation. These data provide an insight into Akt regulation and rational cancer gene therapy by identifying 14-3-3 sigma as a molecular regulator of Akt and as a potential anticancer agent for Akt-activated cancers.

Negative cell cycle regulator 14-3-3σ stabilizes p27 Kip1 by inhibiting the activity of PKB/Akt

The 14-3-3σ (sigma) protein is a human cancer marker downregulated in various tumors, but its function has not been fully established. 14-3-3σ is a negative regulator of cell cycle when overexpressed, but it is not clear whether 14-3-3σ regulates cyclin-dependent kinase inhibitor p27Kip1 to negatively affect cell cycle progression. Protein kinase B/Akt is a crucial regulator of oncogenic signal and can phosphorylate p27Kip1 to enhance p27Kip1degradation, thereby promoting cell growth. Here, we show that 14-3-3σ-mediated cell cycle arrest concurred with p27Kip1 upregulation and Akt inactivation. We show that 14-3-3σ blocks Akt-mediated acceleration of p27Kip1 turnover rate. 14-3-3σ inhibits Akt-mediated p27Kip1 phosphorylation that targets p27Kip1 for nuclear export and degradation. 14-3-3σ inhibits cell survival and tumorigenicity of Akt-activating breast cancer cell. Low expression of 14-3-3σ in human primary breast cancers correlates with cytoplasmic location of p27Kip1. These data provide an insight into 14-3-3σ activity and rational cancer gene therapy by identifying 14-3-3σ as a positive regulator of p27 and as a potential anticancer agent.

Ancillary Techniques in the Followup of Transitional Cell Carcinoma: A Comparison of Cytology, Histology and Deoxyribonucleic Acid Image Analysis Cytometry in 91 Patients

PURPOSE Voided urine and bladder washing cytology are used frequently in the evaluation of transitional cell carcinoma of the bladder. As part of an ongoing investigation we report on the role of deoxyribonucleic acid (DNA) image analysis cytometry as an adjunct to cytology in the followup of patients with transitional cell carcinoma. MATERIALS AND METHODS Urine cytology and image analysis cytometry were performed independently on aliquots of voided urine, catheterized urine or bladder washings from 91 patients with previous or active transitional cell carcinoma of the bladder, and the results were compared to those of concurrent biopsy and clinical followup. RESULTS Of 75 recurrent transitional cell carcinomas 42 were detected by cytology, while 63 and 64 were identified by image analysis cytometry and biopsy, respectively, for a sensitivity of 57, 84 and 85%, respectively. Combined cytology and image analysis cytometry detected 67 recurrences, for an overall sensitivity of 89%. Of 11 cases undetected by concurrent biopsy 9 had abnormal DNA histograms with transitional cell carcinoma at followup and 2 were DNA diploid but with grade 1 transitional cell carcinoma at followup. Of 12 cases undetected by image analysis cytometry 8 were grade 1 and 4 were grade 2 transitional cell carcinoma. CONCLUSIONS Urine cytology and image analysis cytometry detect most recurrent tumors. Their combined use is indicated in the followup of patients with bladder transitional cell carcinoma.

The Mainz Classification of Renal Cell Tumors.

BACKGROUND: Tumors arising from the renal tubular epithelium have variable characteristics and have been subject to a variety of histologic classifications. METHODS: The authors describe the distinct clinical, pathologic, phenotypic, and genotypic features of different types of renal tumors. RESULTS: The Mainz classification is now widely accepted because characteristic genetic alterations have been demonstrated in each tumor type. CONCLUSIONS: The increasing emphasis on utilizing genetic characteristics of specific tumors is reflected by the more widespread use of the Mainz classification for renal cell tumors.

MALDI/SELDI Protein Profiling of Serum for the Identification of Cancer Biomarkers

The ability to visualize the full depth of the serum proteome in a high-throughput manner is a major goal of clinical proteomics. Methodologies, which combine higher throughput with the ability to observe differential protein expression levels, have been applied to this goal. An example of such a system is the coupling of robotic sample processing to matrix-assisted laser desorption time of flight mass spectrometry (MALDI-TOF-MS). Within this paradigm is a modification of MALDI-TOF termed surface-enhanced laser desorption/ionization-TOF (SELDI-TOF). Both conventional MALDI and SELDI have been used to generate protein expression profiles reflective of potential peptide changes in serum. This information can be used to identify proteins, which may enable new diagnostic and therapeutic strategies.

Predictability of PSA failure in prostate cancer by computerized cytometric assessment of tumoral cell proliferation

OBJECTIVES To evaluate the relationship of DNA ploidy and cell proliferation (CP) with Gleason score (GS) and clinical outcome in prostate cancer. METHODS Sixteen patients with benign prostatic hyperplasia (BPH) and 65 patients with prostate cancer classified by GS (four groups: 2 to 4, 5 to 6, 7, and 8 to 10) were studied. All patients with carcinoma underwent prostatectomy and were separated into prostate-specific antigen (PSA) failure and nonfailure groups (failure if PSA 0.1 ng/mL or more three times after surgery). Tumoral CP (Ki-67 inmunostaining and SG2M phase) and DNA ploidy were evaluated by computerized cytometry. RESULTS BPH were diploid with low CP (8% SG2M cells or less). Carcinomas were either diploid with high CP (greater than 8% SG2M cells) or aneuploid. CP was significantly higher (P <0.001) in tumors with GS 7 or greater than in tumors with GS less than 7 (mean percent Ki-67 cells 18.3% versus 7.8%, respectively). PSA failure increased with GS (7.1% in GS 2 to 4, 21% in GS 5 to 6, 28.6% in GS 7, and 50% in GS 8 to 10), as well as with aneuploidy (18.5% in diploid tumors versus 72.7% in aneuploid tumors). Those experiencing PSA failure had significantly higher (P <0.001) CP than those not failing (mean percent Ki-67 cells 24% and mean percent SG2M 30.4% versus 8.7% and 13.5%, respectively). Cox regression analysis showed GS, DNA ploidy, Ki-67, and SG2M to each be univariately prognostic for time to PSA failure; however, Ki-67 and SG2M were more highly significant (P <0.0001 for both) than GS (P = 0.007) or DNA ploidy (P = 0.002). After adjusting for either SG2M or Ki-67 measures of CP, neither ploidy nor GS contained additional prognostic value. CONCLUSIONS Tumor CP and DNA ploidy can be reliably determined in prostate cancer by computerized cytometry. On the basis of our preliminary results, CP correlates well with GS and predicts PSA failure better than DNA ploidy or GS.

Multilocular prostatic cystadenoma with high-grade prostatic intraepithelial neoplasia.

Multilocular prostatic cystadenoma is a rarely encountered neoplasm located in the midline between the bladder and rectum that is either attached to the prostate by a pedicle or separate from the prostate entirely. Histologically and immunohistochemically these lesions resemble benign prostate tissue. We report the first case of this entity for which multifocal high-grade prostatic intraepithelial neoplasia (PIN) is identified. Conceptually, the finding of high-grade PIN in multilocular prostatic cystadenomas provides further evidence that these lesions are fully analogous to the prostate gland not only in their morphology and immunohistochemistry but also in their predilection for the same diseases.