BIAS OF SELDI-TOF TECHNOLOGY TOWARD HIGH-ABUNDANCE MOLECULES
The current method of performing SELDI-TOF experiments with unfractionated serum includes exposure of serum to the protein chip, washing, and then identification of the immobilized molecules by using MALDI-TOF instrumentation. The solid phases currently in use (mentioned earlier) are not specific for any type of protein. Because serum contains a tremendous array of extremely high-abundance (e.g. albumin) and very-low-abundance molecules (range of concentrations vary by a factor of 106- to 109-fold) (45), it will be highly unlikely that the most informative, low-abundance molecules will be able to immobilize on such chips. Simply, they will likely be competed out by high-abundance, noninformative molecules. For example, in serum, the PSA concentration in healthy males is 1 µg/liter, whereas the total protein concentration is in the order of 80,000,000 µg/liter. When proteins are exposed to the chip, each PSA molecule (or other molecules of similar abundance) will encounter competition for binding to the same matrix by millions of irrelevant molecules. It would thus seem very unlikely that molecules with very low abundance will ever be detected by this method. The experiments to prove or disprove these proposals have been previously outlined by this author in a separate editorial, but to my knowledge they have not as yet been reported (46).
A previous report by Wright et al. claimed that four classic prostatic biomarkers, including free and complexed PSA, could be detected by mass spectrometry in various biological fluids and tissue extracts, including seminal plasma, prostatic extracts, and serum (47). However, the masses assigned to free or complexed PSA may have originated from other molecules with a similar molecular mass. Furthermore, the presence of other molecules, such as salts, could cause a mass shift, thus complicating the interpretation further. These authors, in their efforts to show a quantitative relationship between peak area and PSA concentrations, constructed linear calibration curves but at PSA concentrations between 1,000 and 50,000 µg/liter. Such concentrations are rarely or never seen in clinical practice, even in sera from patients with highly metastatic prostate cancer. On the same point, other authors reported prostate-specific membrane antigen (PSMA) concentrations in serum (this is another prostatic-specific molecule) by using a SELDI-TOF approach in combination with an immobilized antibody. The reported concentrations of PSMA in serum (100–500 µg/liter; 500 times higher than the secreted protein PSA) are surprisingly high and need to be validated by ELISA-type methodologies, given that this molecule is a membrane-bound protein (48).
I have compiled a list of SELDI-TOF-identified molecules in serum that are thought to be discriminatory between normal stage and cancer (Table IV). Clearly, these candidate serum biomarkers are very-high-abundance molecules known to be produced mainly by the liver.
For example, Zhang et al. (49) identified three discriminatory peaks in ovarian cancer: apolipoprotein A1, transthyretin (pre-albumin) fragment, and inter--trypsin inhibitor. Ye et al. discovered haptoglobin- subunit for ovarian cancer (50), and Hlavaty et al. discovered vitamin D-binding protein for prostate cancer (51). More recently, Cho et al. identified serum amyloid A protein for nasopharyngeal carcinoma (52). Table IV presents the comparative serum concentrations of these putative tumor markers and of classical tumor markers, such as -fetoprotein and PSA.
A number of the "new" tumor biomarkers discovered by SELDI-TOF technology were, in fact, originally identified more than 30 years ago by classical techniques (e.g. haptoglobin- subunit for ovarian cancer) (53) but were deemed useless for clinical diagnosis because of their low sensitivity and specificity (54, 55). Just to illustrate this point further, I performed a MEDLINE search using the keywords "haptoglobin" and "cancer" and identified 571 papers published from 1965 to 2003. Haptoglobin was reported since 1966 to be elevated in the following malignancies: leukemias, Hodgkin’s disease, Burkitt’s lymphoma, multiple myeloma, neuroblastoma, melanoma, glioma, and cancers of the cervix, genitals, stomach, breast, liver, kidney, ovaries, lung, endometrium, colon, prostate, gallbladder, bladder, head and neck, brain, and larynx. The same comments applies to serum amyloid A protein (52). It is clear that haptoglobin- subunit or other acute-phase reactants are not specific cancer biomarkers.