Cancer Biomarker Discovery via Low Molecular Weight Serum Profiling—Are We Following Circular Paths?

The rigors of attaining reproducible protein identifications from complex biological matrices have recently been described as the ascent of a “mountainous road” that must surmount a series of methodological, technical, and analytical barriers to attain reliable results (1). From the perspective of biomarker discovery, the difficulty of this trail, and the attendant requisite level of expertise, is greatest when broad discovery work flows are used but is lessened substantially if targeted strategies can be used. Taking broad poetic license to portray this visual image within the context of the American westward expansion, one can envision a process by which teams must leave the comfort of the gentle plains (the proof-of-concept phase) to scale the foothills and peaks that lie on the trail to clinical utility. There will be much debate on the choosing of the best path forward. With this simile in mind (and to our point of view), we still see, as we suggested a few years ago, that many advocates of the use of mass spectrometry (MS)1 for profiling the so-called low molecular weight fragmentome (LMWF) remain circling on the plains, retracing the paths of evidence laid down decades before that had revealed the prevalence of dysregulated hemostasis in malignant disease (2). The genesis of what has been referred to as the “SELDI fiasco” (3) traces back to the early success and attendant hyperbole associated with the apparent differentiation of ovarian cancer patients from their unaffected controls by a pattern of uncharacterized peaks presented in the low-mass region of native serum SELDI analyses (4). Although the discriminatory power of these results was ultimately attributed to methodological bias (5), the concept of the use of biomolecule patterns as disease-specific identifiers had been proposed, and the need for component identification had been disputed. Qualitative data obtained by liquid chromatography coupled with tandem MS (LC-MS/MS) (6) subsequently revealed the LMW plasma proteome to consist largely of proteolytic fragments of abundant blood proteins associated with coagulation and the complement cascade, most of which are now known to be produced ex vivo. Skeptics in the field questioned the suitability of these approaches to produce novel insights into disease, given their sensitivity to preanalytical influences and given the prior knowledge of the prevalence of hemostatic dysregulation in oncology (2). In contrast, some advocates of LMWF profiling, having observed similar findings in their own hands, invoked the presence of tumor-specific exopeptidases to account for the apparent specificity of ex vivo–dependent disease patterns and, in a welcome break from the field in general, have taken the appropriate steps toward establishing a rigorous assay platform to carry this effort forward (7). Regardless of opinion, these and other data offer compelling evidence that the serum LMWF, when probed by direct analyses of unfractionated materials, is at a minimum confounded by and at worst perhaps limited to the detritus of abundant blood proteins. We suggest the successes described to date may be due to dysregulated hemostasis—often overlaid on an acute-phase response—but are unlikely to be due to anything more. In light of the daunting concentration range of serum components, the limited dynamic range of MALDI analyses (8), and the dramatic impact of ex vivo proteolysis, this conclusion is the simplest explanation of the observed phenomena (i.e., the rule of Occam’s razor is satisfied). With the annotated features from a study of both plasma and serum (9), Fig. 1 presents a glimpse into the potential peptide complexity of the blood LMWF over the LC-MS/MS–tractable mass range. Given the usual caveats associated with sequencing by tandem MS (i.e., not all ions yield quality spectra, and not all quality spectra can be easily correlated in a database search) and the likelihood that the identified peptide ladders represent facile components of their “family trees,” it is reasonable to suggest there are multiple components at every nominal mass in a MALDI spectrum of an unfractionated sample, be it plasma or serum. The shadow cast by these fragments of abundant proteins, >90% of which represent the top 75 and top 125 proteins in plasma and serum, respectively (10), obscures the likelihood of detecting tumor-derived peptides in a MALDI spectrum. Although the various qualitative assessments of native blood fluids performed since 2005 have generally failed to uncover tumor-specific peptides, they have revealed the major impacts that preanalytical and technical variables have at all stages, from the point of sample collection through the final data analysis. The important point here is that the disease associations of dysregulated hemostasis are known and are measured as part of regular clinical care. What can easily be measured with current clinical assays becomes more complex to analyze at the level of the LMWF. Mass distribution of annotated native peptides observed in human plasma and serum [Bakun et al. (9)]. Peptides derived from proteins represented by at least 2 unique peptide ions are displayed within the mass range of 700–4000 Da. The peptides annotated in the plasma analyses are derived exclusively from blood proteins with 90% of these peptides representing the top 75 proteins by relative abundance [Hortin et al. (10)]. More than 90% of the serum peptides are derived from the top 125 serum proteins, whereas <1% of all peptides were attributed to non-serum nor platelet-derived proteins. The cycle of rediscovery can be seen in the frequent observations of seemingly promiscuous LMW features across a number of studies. One of these features, a temporally sensitive biomarker of approximately 5909 nominal mass, has repeatedly been identified as an internal fragment of the fibrinogen α chain spanning residues 576–629 (FIBA 5909). Although the fragment is directly correlated with serum coagulation in healthy individuals (2), its presence in samples from diseased individuals is likely due to the same mechanism. Despite numerous reports of its identification and correlation with the coagulation process, FIBA 5909 continues to be rediscovered and reported as “uncharacterized” (11). Remaining current with the biomarker literature has grown increasingly difficult over the years, with the growth of the field and the advent of new journals sharing the responsibility. Consequently, reporting of this particular observation is likely to continue until the identification of discriminating features is required before their publication. Additionally, the process of sample collection initiates profound changes in the LMWF through the initiation of proteolytic cascades and the activation of blood cells and platelets, the complexity and regulation of which elude full understanding. The lack of perfect knowledge, however, should not preclude the recognition that diseases affecting platelet biology, such as many malignant states, are likely to yield discriminating features in native MS analyses that are hallmarks of platelet activation (e.g., platelet factor 4, pro–platelet basic protein precursor) (12)(13). The interpretation of results that implicate the involvement of platelet-derived factors is incomplete without the patient’s platelet count and morphology. Early critics of the Ciphergen Biosystems ProteinChip Reader (PBS-II) reflected on the low resolution and low mass accuracy of this relatively unsophisticated MALDI-TOF instrument. Anecdotally, it was often expressed that one could do better with a “real” mass spectrometer. Although the benefits of enhanced mass resolution, accuracy, and stability are indisputable and the value of off-chip sample processing has been demonstrated (14), these features are fine points compared with the magnitude of the effect induced by the underlying biology. As has been reviewed recently (15), 45% of the peaks observed in differential analyses of samples from case–control studies that targeted breast cancer on an “advanced” platform represented rediscoveries of prior findings, with the FIBA 5909 peptide being among the most significantly correlated. Similarly, the pairwise recapitulation of observations obtained with the low-performance PBS-II instrument and with high-value instrumentation (16) regarding the prevalence of abundant protein degradants (including FIBA 5909) in sera from patients with head and neck squamous cell carcinoma suggests that little new biology is likely to be revealed. The reliability of these platforms is undoubtedly superior, and the tandem sequencing capabilities will surely prove valuable. But will the outcomes differ? Time will tell, but the key benchmark of progress will be evident when feature identification becomes the accepted practice—a task that would be accelerated in many cases if the compendium of identified peptides were to be curated in a centralized database accessible through tools such as TagIdent (17). Circular paths are often trod with respect to the analysis of small acute-phase proteins such as serum amyloid A, as is evident in its discovery as a putative therapeutic response marker in the treatment of non–small-cell lung cancer (18)(19). Although inconsistently identified by the research team (known in 2007 but unknown in 2009), the profile of serum amyloid A is consistent and unmistakable, with expression levels inversely correlated with the response to treatment (i.e., survival). Its prognostic value has also been consistent and unmistakable across decades of investigation (20). The recent US Food and Drug Administration approval of the OVA1 screening test (Vermillion) speaks to the value of evaluating serum amyloid A and similar proteins, because 2 of the 4 proteins (transthyretin and transferrin) evaluated in combination with cancer antigen 125 are acute-phase reactants, although they are negative responders in this example (21). In view of this acceptance of “the pattern is the biomarker” model, it follows that any examination of the serum LMWF is incomplete without the parallel assessment of the acute-phase reactants. Combined with our previous recommendations regarding coagulation and platelet status, all of which are components of standard clinical practice, systematic evaluation of these cellular and molecular components should expand our understanding of the underlying biology and better determine, perhaps more rapidly, the utility of LMW profiling. This commentary is not meant to disparage the efforts of these research groups, and we recognize the difficulty of attaining a comprehensive coverage of the field in today’s information-rich environment. We acknowledge our own limitations in this regard with respect to the omission of references to efforts with proximal fluids and tissues, which are more likely to yield disease insights than serum profiling, or with respect to the emerging recognition of the potential value of isoform variation and posttranslational modifications, which are all beyond the scope of this commentary. This criticism is intended to guide readers toward a fact-based recognition of the inherent limitations of MALDI/SELDI profiling of the LMWF for biomarker discovery and to serve as an instrument to encourage others to strike out on their own road. In an offhanded fashion, the emerging trend toward publication of negative data, which effectively closes out years of preliminary promise, is evidence that the field can break the cycle and pursue other paths (22)(23). At the same time, one must wonder if the recent report of a LMW profile for the early detection of breast cancer does not feel like déjà vu (24). Nonstandard abbreviations: MS, mass spectrometry; LMWF, low molecular weight fragmentome; LC-MS/MS, liquid chromatography–tandem MS; FIBA 5909, 5909-Da internal fragment of the fibrinogen α chain spanning residues 576–629.