Protein Profiling of Pleural Effusions to Identify Malignant Pleural Mesothelioma Using SELDI-TOF MS

Diagnosis of malignant pleural mesothelioma (MM) is limited. Novel proteomic technologies can be utilized to discover changes in expression of pleural proteins that might have diagnostic value. The objective of this study was to detect protein profiles that could be used to identify malignant pleural mesothelioma with surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Pleural effusions were collected from patients with confirmed mesothelioma (n = 41) and from patients with effusions due to other causes ([n = 48] cancerous and non-cancerous). Samples were fractionated using anion exchange chromatography and bound to different types of ProteinChip array surfaces. All samples were also subjected to other commercially available immunoassays (human epididymes protein 4 [HE4], osteopontin [OPN], soluble mesothelin-related proteins [SMRP], and the cytokeratin 19 fragment [CYFRA 21–1]). Peak intensity data obtained by SELDI-TOF were subjected to classification algorithms in order to identify potential classifier peaks. A protein peak at m/z 6614 was characterized as apolipoprotein (Apo) CI. In this setting, the sensitivity and specificity of this potential biomarker was 76 % and 69 %, respectively. The area under the receiver operating characteristic curve (AUC) for Apo CI was 0.755, thereby outperforming OPN, HE4, and CYFRA 21–1. SMRP performed best with an AUC of 0.860 with a sensitivity of 83% and specificity of 74%. Our study validates the use of SMRP as a diagnostic marker for pleural mesothelioma and furthermore suggests that Apo CI levels could be used in the future to discriminate pleural mesothelioma from other causes of exudates.

A Unique Biomarker Is Associated with the Prevalence of Functional Heparin Induced Thrombocytopenia (HIT) Associated Antibodies: Results from Proteomic (ProteinChip Array) Profiling of ELISA Positive HIT Plasma Samples.

Heparin induced thrombocytopenia (HIT) represents a complex immunopathologic syndrome resulting in the activation of coagulation, inflammatory and cellular processes, which contribute to the overall pathogenesis. Protease mediated transformation of endogenous proteins resulting in the generation of certain mediators and specific cleavage products may be used to understand the pathogenesis of this syndrome. It was hypothesized that unique biomarkers may be generated in patients who develop HIT antibodies upon exposure to heparins. The identification of unique biomarkers and their prevalence in heparin exposed patients may provide an additional parameter in the prognosis of this syndrome. To profile the proteomic biomarkers in serum, specimens were selected from archived sera that had been referred to Loyola University Medical Center for the quantitation of HIT antibodies and 14C Serotonin Release Assay (SRA). All of these specimens were positive in the GTI PF4 Enhanced ELISA for the presence of anti-heparin platelet factor 4 antibodies with a broad range of antibody titers absorbance range (0.4 – 2.5). Eleven of these specimens were positive in the SRA. All of these samples were proteomic profiled utilizing a ProteinChip Array method using a Gold (AU) Chip in the molecular weight range of 0 – 150 kDa. The normal sera samples (n = 40) were also analyzed simultaneously in a crossover fashion along with the patients’ specimens. Surface enhanced laser desorption/ionization (SELDI) technique utilizing Ciphergen PBS II (Fremont, CA) was used for mass profiling. Proteomic profiling is widely used to identify unique biomarkers in various hemotologic disorders. Although electrophoretic methods identify biomarkers at molecular weights ≥ 25 kDa, the SELDI technique provides a complete spectrum in the lower molecular weight range. The ProteinChip Software for mass profiling was used to analyze the data. Proteins such as albumin were used as control proteins throughout the study. Of the 54 HIT samples profiled, 36 showed a unique biomarker peak at 11.9 kDa (67%) whereas none of the normal sera (n = 40) showed this peak. All of the SRA positive samples also showed the 11.9 kDa biomarker peak. Several biomarker peaks in the range of 5 – 10 kDa were present in the HIT positive sera suggesting increased proteolysis. While no other unique biomarkers were obvious in the HIT samples, the relative proportion of some of the biomarker peaks in the range of 15 – 20 kDa were differentiable in the HIT group. These studies demonstrate that HIT patients’ sera exhibit a unique biomarker profile, which is distinguishable from normal serum samples. Combined usage of the ProteinChip Array analysis of the HIT patients’ sera with other diagnostic methods may further enhance the diagnostic process for this syndrome. Additional molecular characterization of these biomarkers will also help in the understanding of the pathogenesis of HIT with particular reference to the protease activation processes.

ProteinChip Array Profiling and Markers of Inflammation and Thrombin Generation in Plasma Samples from Lung Cancer Patients and Their Modulation by Chemotherapy with or without Warfarin Anticoagulation.

While the increased prevalence of venous thromboembolic events (VTE) is fully recognized in lung cancer, its pathogenesis is not fully understood. Profiling of surrogate markers of thrombosis and inflammation provides an opportunity to understand the pathogenesis of VTE. More recently, ProteinChip Array technology has provided a novel approach to profile unique biomarkers in cancer patients. Protease activation in these patients results in the generation of unique biomarkers, which can be identified and monitored during the course of patient management. In a prospective, randomized, controlled study patients with inoperable lung cancer (n=100) were randomized to receive A) chemotherapy, radiation and warfarin (INR 1.5–2.5) or B) chemotherapy, radiation without warfarin (n=50). Blood samples were drawn prior to and after the second treatment cycle in both groups A and B. All samples were also analyzed for such inflammation markers as the tumor necrosis factor alpha (TNFa), CD 40 ligand (CD 40L), C-reactive protein (CRP), interleukin 1 beta (IL-1B), asymmetric dimethylarginine (ADMA) and nitric oxide (NO). In addition, prothrombin fragment F1.2 (F1.2), thrombin antithrombin complex (TAT) and functional microparticles were also measured. Proteomic profiling is widely used to identify unique biomarkers in various hemotologic disorders. Albumin was used as control proteins throughout the study. The baseline blood levels of the inflammatory markers and coagulation activation marker were increased in both the warfarin treated and control group. After the second treatment cycle, the warfarin treated group exhibited varying levels of decrease in all of the surrogate markers of coagulation activation and inflammation (13 – 15%). On the other hand, none of the warfarin treated group showed a marked increase in the TNFα, CD40L, NO, F1.2 and TAT (18–30%). No change in the IL-1B was noted in this group. However, CRP levels were markedly reduced (46%). Most of the baseline samples in both groups showed a unique biomarker peak at 11.9 kDa. The prevalence of this unique biomarker peak was decreased after the second cycle of chemotherapy and after the final course of chemotherapy, it was totally diminished. No significant differences in the down regulation of this unique biomarker were obvious in both groups. Since inflammatory markers showed a decrease (13–50%) in the warfarin treated group, whereas the non-warfarin treated group exhibited an increase (18–30%) in all markers except IL-1B, CRP and ADMA, anticoagulation down regulates these mediators. Markers of thrombin generation were also down regulated in the warfarin treated group. The identification of the unique biomarker at 11.9 kDa in both groups and its gradual down regulation eventually leading to diminution in both groups, suggests that chemotherapy and radiation treatment appear to regulate this biomarker. The levels of various inflammatory markers are upregulated in lung cancer suggesting a pathogenic role of this process in lung cancer. Warfarin down regulated the inflammatory process in contrast to the non-warfarin treated group. However, the unique biomarker at 11.9 kDa appears to be regulated by chemotherapy and radiation. The clinical relevance of these observations requires additional investigations.

Upregulation of Microparticles and CD40 Ligand in Antiphospholipid Syndrome: Potential Implication in Inflammatory Responses and Thrombosis.

Antiphospholipid syndrome (APS) represents a complex pathphysiologic state where vascular endothelium, platelet activation, coagulation and inflammatory processes are contributory to the overall outcome. Cellular apoptosis and immune mediated fragmentation results in the generation of microparticles (MP). Platelet associated CD40 Ligand contributes to the inflammatory complications, which may be further augmented by MP. To test the hypothesis that both the MP and CD40 ligand are increased in patients with APS, 90 samples received at Hines VA/Loyola Medical Center laboratories for antiphospholipid screening were profiled for MP utilizing a functional method and CD40 ligand employing an ELISA method (R&D, Minneapolis, MN). ProteinChip Array analysis was also carried out for biomarker profiling to identify a previously described inflammatory biomarker at 11.9 kDa utilizing surface enhanced laser desorption/ionization (SELDI). Fifty normal healthy male and female plasma samples were used as controls for comparative purposes. Of the 90 samples screened, 30 were found to be positive for the antiphospholipid antibody (APA) titer using an ELISA method (American Diagnostica, Stamford, CT). All of the 30 positive samples have elevated DRVVT; however, wide variations in the APTT were noted. The remainder of the 60 samples, which were negative for APA, also showed variability in the DRVVT and APTT values. In contrast, the DRVVT and APTT values in the normal were within the expected range. None of the normal samples were positive for the APA antibody. Most strikingly, the CD40 ligand in the samples positive for APA titer were markedly higher (480 ± 230 pg/ml) than the 60 which were negative (180 ± 80 pg/ml). The normal values were much lower (90 ± 30 pg/ml). The MP titer in the APA positive samples was also much higher (34 ± 14 nM) in contrast to the APA negative samples (18 ± 12 nM). The normal levels were even lower (9 ± 4 nM). In the ProteinChip Array analysis, the APA positive samples showed a higher prevalence of the unique biomarker at 11.9 kDa. This unique biomarker was absent in the normal human plasma. These findings clearly suggest that APA represents a complex syndrome where hemostatic activation involving the formation of the MP, upregulation of inflammatory process as evident by the generation of CD40 ligand and protease activation resulting in the formation of unique biomarkers contribute to its overall pathophysiologic outcome. Sequential profiling of these parameters may be helpful in the risk stratification of these patients.

ProteinChip Array Profiling for Identification of Disease- and Chemotherapy-Associated Biomarkers of Nasopharyngeal Carcinoma

Background: We previously used ProteinChip array profiling analysis to discover a serum biomarker associated with nasopharyngeal carcinoma (NPC). In this study, we used the same method to examine other biomarkers associated with NPC and response to chemotherapy (CT) in NPC patients. Methods: We performed ProteinChip array analysis in 209 serum samples from 66 relapsed patients before and after salvage CT with gemcitabine and cisplatin or etoposide and cisplatin combinations, 11 patients in remission, and 35 healthy individuals. Intensities of the biomarker peaks were correlated with CT response of the patients and other clinical parameters. Results: We discovered 13 candidate biomarkers associated with different clinical parameters. Two biomarkers (2803 and 3953 Da) were significantly increased in patients compared with controls at all stages of disease. Analysis of pre- and post-CT paired serum samples revealed 7 biomarkers correlated with impact of CT. Of these 7 biomarkers, 2 (2509 and 2756 Da) were significantly increased and 5 (7588, 7659, 7765, 7843, and 8372 Da) were significantly decreased post-CT in either 1 or both CT cohorts. Four biomarkers from pre-CT sera were correlated with CT response, with 3 (2950, 13 510, and 14 855 Da) being significantly decreased and 1 (6701 Da) significantly increased in patients who did not respond to CT. Tandem mass spectrometric sequencing and/or immunoaffinity capture assay identified the 3953 Da biomarker as a fragment of interα-trypsin inhibitor precursor and 7765 Da biomarker as platelet factor-4. Conclusions: Treatment-associated serum biomarkers found might serve to triage NPC patients for appropriate CT treatment.