Practical Clinical Applications

In addition to the potential diagnostic power of a given MS pattern, the advantages of delineating the exact relationship between the various proteins and the disease are seemingly infinite. The identified peptides and proteins can be used for screening, diagnosis, staging, prognosis, monitoring treatment response, or detection of tumor recurrence [73]. Proteomic technology has relevance to every field of medicine and science, making a complete review of the current applications beyond the scope of this chapter. Instead, a few remarkable examples demonstrating the breadth of possibilities are presented, including cancer and infectious diseases [reviewed in 49, 74, 75]. Other topics of interest that are not covered include evaluating drug toxic-ities [76, 77]; profiling of neuropsychiatric disorders such as schizophrenia, Alzheimer disease, and Parkinson disease [reviewed in 78]; immunopheno-typing of leukemias with a long-term goal of monitoring minimal residual disease [79], and characterizing human spermatozoa surface antigens in relation to immunological infertility [25].

8.1. Tumor Markers

Because 2D-PAGE and MS patterns can be used to differentiate between healthy and cancer patients, the proteins in these spectra contain tumor markers. If they could be isolated and identified, they would be useful for innumerable purposes, most notably for early detection. If a tumor marker can warn of the presence of disease at an early stage, before the cancer can be visualized using imaging studies, this would increase the chances of treating at a curative stage.

Tumor antigens can be classified as tumor-specific antigens if they are exclusive to a particular type of cancer cell, or tumor-associated antigens if they are not unique to malignant cells, but are expressed to a significantly greater extent in tumor relative to normal cells. Tumor-associated antigens and tumor-specific antigens include mutated oncogene proteins (p53, c-myc) [80, 81], embryonic proteins (alpha-fetal protein, carcinoembryonic antigen) [82] peptides from immunoprivileged sites (cancer-testis antigens) [83], and overexpressed proteins (HER2/neu) [84-86].

Many tumor markers have been identified in recent years since the improvements in proteomic technologies. For example, one study analyzed human serum with 2D-PAGE and MALDI-TOF-MS to identify a distinct repertoire of autoantibodies associated with hepatocellular carcinoma. These proteins differentiated the cancer patients from those chronically infected with hepatitis B or C, which constitute a high-risk group for developing hepatocellular carcinoma. Thus, proteomic-based technology was used to identify a set of four proteins that may have utility in early diagnosis of hepatocellular carcinoma [87].

A different study used 2D-PAGE and MS to detect proteins from lung adenocarcinoma tissue associated with patient survival. Importantly, protein profiles were used to predict survival of stage I tumor patients. The current standard of care for these patients is surgical resection alone. Therefore, if high-risk stage I individuals could be singled out, they could receive more aggressive adjuvant therapy, which could increase survival rates [88].

Treatment regimens are often based on the predicted clinical outcome; thus, physicians differentially administer aggressive or conservative therapies. This is especially true for prostate cancer, where some men develop advanced disease at a young age and others are merely under observation for prolonged periods of time. To better characterize the immune response in prostate cancer patients, a study was conducted using HPLC and protein microarrays. 2D liquid chromatography was used to separate proteins from the prostate cancer cell line LNCaP into 1760 fractions. These fractions were spotted onto microarrays, which were then incubated with serum samples from men with prostate cancer and male controls. A bioinformatics-based decision tree with two levels of partitioning classified the samples with 98% accuracy as either prostate cancer or control. These results indicated that patterns of immune recognition generated from microarrays of fractionated proteins could be used for prostate cancer diagnosis [89].

Although efforts are underway to identify markers in serum and prostate tissue, the question arose as to whether metastatic prostate cancer cell lines accurately represent in vivo disease. It was found that in vitro cell cultures (LnCaP and PC3) shared less than 20% of proteins when compared to in vivo LCM procured malignant prostate cancer. 2D-PAGE protein profiles were used to compare normal and malignant cells to immortalized cells from the same patient. Protein expression patterns were dramatically altered when cells were grown in culture and immortalized; most notably, a loss of prostate specific antigen expression was observed [18]. Thus, caution must be used when working with immortalized cell lines to discover potential disease markers.

8.2. Disease-Related Applications

Current diagnostic methods rely on invasive procedures, such as biopsies, to accurately assess many conditions. For many disorders, visualization of the pathology via imaging modalities is only possible at an advanced stage. One of the main goals of proteomics is to use readily accessible body fluids to make an accurate diagnosis earlier in the course of the disease. One issue is whether all diseases have associated cells that secrete or shed markers that can be reliably detected through serum or urine profiling, thus obviating the need to perform more invasive procedures. It is still not known which pathologies can be diagnosed by analyzing body fluids, and which ones can be detected only by examining the tissues themselves.

Differential diagnosis of graft-versus-host disease currently depends on organ biopsy to distinguish it from other common complications associated with transplantation. As a means of avoiding repeated biopsies, one group analyzed the urine of patients after hematopoietic stem cell transplantation to generate a peptide pattern that could be used to diagnose graft-versus-host disease. Capillary electrophoresis and electrospray ionization-TOF-MS were used to evaluate the protein patterns in urine from 40 posttransplant patients and compared them to those of five patients with sepsis. Peptides present or absent with an absolute difference of more than 50% or with a more than 10-fold difference in the MS signal intensity between the two groups were accepted as significant disease markers. Sixteen graft-versus-host-disease-specific and 13 sepsis-specific peptides were identified, showing that this technology may be used as a powerful diagnostic tool. Early and accurate identification of patients developing complications after transplantation can lead to more timely and appropriate therapeutic interventions, ultimately translating into reduced morbidity [90].

Since 1986, scientists had been trying to identify the CD8 antiviral factor that was found in greater than normal amounts in certain HIV-1-infected individuals [91]. The elusive CD8 antiviral factor was of tremendous clinical interest because it conferred immunological stability, characterizing this group as long-term nonprogressors. The breakthrough discovery of CD8 antiviral factor being a-defensin 1,2, and 3 was made possible with the use of SELDI-TOF-MS and searching through protein databases. Only with the help of modern proteomic technology was the extensive 16-year search effectively ended with a greater understanding of the soluble peptide factors suppressing HIV-1 replication [92].

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