Validation of SELDI-TOF MS serum protein profiles for renal cell carcinoma in new populations

Abstract

Currently, no suitable biomarker for the early detection or follow-up of renal cell carcinoma (RCC) is available. We aimed to validate previously reported potential serum biomarkers for RCC obtained with Surface Enhanced Laser Desorption Ionisation-Time of Flight Mass Spectrometry (SELDI-TOF MS) in our laboratory using distinct patient populations. Two sets of sera from RCC patients and healthy controls (HC) were gathered from different institutes and analysed according to published procedures. The first set (40 RCC, 32 HC) consisted of mainly presurgery samples from patients with disease stages I-IV. The second set (26 RCC, 27 HC) were mostly sera from patients with stage-IV disease, drawn after nephrectomy. Only the increased expression of the previously found serum amyloid-a (SAA) peak cluster could be validated in a similar RCC patient subset in both our populations in two independent analyses. It was seen both in earlyand late-stage disease and in pre-and postsurgery samples. These results were also confirmed by ELISA. Other previously identified biomarker candidates (mass-to-charge ratio's (m/z) 3900, 4107, 4153, 5352 and 5987) proved difficult to reproduce upon duplicate analysis. Modification of the analytical protocol for these markers resulted in their detection, but we did not achieve satisfactory classification of patients and controls with these alleged biomarkers in any of our two sample sets. Instead, two new peaks (m/z 4289 and 8151) were identified with better performance (sensitivity and specificity B65-90%) for separating patients from controls in the first sample set. Concluding, only the SAA peak cluster was validated as a robust RCC biomarker candidate, which is present in a specific subset of these patients, regardless of disease stage or nephrectomy status. In addition, two new peaks were seen which might prove useful as biomarkers, provided these are validated in new populations. Renal cell carcinoma (RCC) is difficult to diagnose at early stages due to a lack of clear clinical symptoms. When symptoms do occur, about 30% of patients already have metastatic disease. In addition, a similar part of patients with resection of localised disease will have a recurrence. 1 Therefore, a need remains for reliable markers for diagnosis and follow-up of RCC, preferably in easy-accessible body fluids. Surface Enhanced Laser Desorption Ionisation-Time of Flight Mass Spectrometry (SELDI-TOF MS) 2,3 is being increasingly used to search for new and better tumour markers in (serum) protein profiles, for example, for ovarian, 4 breast, 5 prostate 6 and colorectal cancer. 7 Its appeal lies in the ease with which a multitude of samples can be analysed with a minimum of sample preparation in a single SELDI-TOF MS analysis. Indeed, SELDI-TOF MS has also been performed to identify biomarker proteins for early detection of RCC. Two studies describe protein profiling of serum with SELDI-TOF MS. 8,9 Tolson et al 8 reported a peak cluster at a mass-to-charge ratio (m/z) of approximately 11 000 from serum amyloid a-...

Serum proteomics and disease-specific biomarkers of patients with advanced gastric cancer

“…Finally, the chips were rinsed with deionised water, air dried and finished with two 1-µl SPA applications to the sample spots. the reproducibility of the applied methodology was previously validated by our group (11).…”

“…Data collection was optimized for detection of discriminating peaks, resulting in an average of 65 laser shots per spectrum at laser intensity 150, detector sensitivity 8 and laser focusing at 3,000 Da. M/z values for the detected proteins were calibrated externally with a standard peptide mixture (Biorad laboratories) containing vasopressin (1,084.3 Da), somatostatin (1,637.9 Da), dynorphine (2,147.5 Da), ActH (2,933.5 Da), insulin β-chain (bovine; 3,495.5 Da), insulin (human recombinant; 5,807.7 Da) and hirudin (7,033.6 Da) (11).…”

“…Although some m/z values were difficult to reproduce, the increased expression of the previously identified serum amyloid-α (sAA) peak cluster was validated in different patient populations (11).…”

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“…A special mass spectrometric technique for protein profiling studies is the ProteinChip-based SELDI TOF MS (SELDI-TOF, BioRad, Hercules, CA, USA). These techniques were widely used in the recent years in cancer research [73][74][75]. Due to their high sensitivity, these chips are very suitable for the analysis of small volumes such as ocular fluids.…”

Differencial proteome of clear-cell renal cell carcinoma (ccRCC) tissues

“…Therefore, in order to ensure that a putative biomarker is robust enough for detection in different laboratories, validation with an independent sample set originating from a different institute is essential (9).…”