Alex J. Rai

Three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer

Early detection remains the most promising approach to improve long-term survival of patients with ovarian cancer. In a five-center case-control study, serum proteomic expressions were analyzed on 153 patients with invasive epithelial ovarian cancer, 42 with other ovarian cancers, 166 with benign pelvic masses, and 142 healthy women. Data from patients with early stage ovarian cancer and healthy women at two centers were analyzed independently and the results cross-validated to discover potential biomarkers. The results were validated using the samples from two of the remaining centers. After protein identification, biomarkers for which an immunoassay was available were tested on samples from the fifth center, which included 41 healthy women, 41 patients with ovarian cancer, and 20 each with breast, colon, and prostate cancers. Three biomarkers were identified as follows: (a) apolipoprotein A1 (down-regulated in cancer); (b) a truncated form of transthyretin (down-regulated); and (c) a cleavage fragment of inter-α-trypsin inhibitor heavy chain H4 (up-regulated). In independent validation to detect early stage invasive epithelial ovarian cancer from healthy controls, the sensitivity of a multivariate model combining the three biomarkers and CA125 [74% (95% CI, 52–90%)] was higher than that of CA125 alone [65% (95% CI, 43–84%)] at a matched specificity of 97% (95% CI, 89–100%). When compared at a fixed sensitivity of 83% (95% CI, 61–95%), the specificity of the model [94% (95% CI, 85–98%)] was significantly better than that of CA125 alone [52% (95% CI, 39–65%)]. These biomarkers demonstrated the potential to improve the detection of early stage ovarian cancer.

Proteomic Approaches to Tumor Marker Discovery

CONTEXT Current tumor markers for ovarian cancer still lack adequate sensitivity and specificity to be applicable in large populations. High-throughput proteomic profiling and bioinformatics tools allow for the rapid screening of a large number of potential biomarkers in serum, plasma, or other body fluids. OBJECTIVE To determine whether protein profiles of plasma can be used to identify potential biomarkers that improve the detection of ovarian cancer. DESIGN We analyzed plasma samples that had been collected between 1998 and 2001 from patients with sporadic ovarian serous neoplasms before tumor resection at various International Federation of Gynecology and Obstetrics stages (stage I [n = 11], stage II [n = 3], and stage III [n = 29]) and from women without known neoplastic disease (n = 38) using proteomic profiling and bioinformatics. We compared results between the patients with and without cancer and evaluated their discriminatory performance against that of the cancer antigen 125 (CA125) tumor marker. RESULTS We selected 7 biomarkers based on their collective contribution to the separation of the 2 patient groups. Among them, we further purified and subsequently identified 3 biomarkers. Individually, the biomarkers did not perform better than CA125. However, a combination of 4 of the biomarkers significantly improved performance (P < or =.001). The new biomarkers were complementary to CA125. At a fixed specificity of 94%, an index combining 2 of the biomarkers and CA125 achieves a sensitivity of 94% (95% confidence interval, 85%-100.0%) in contrast to a sensitivity of 81% (95% confidence interval, 68%-95%) for CA125 alone. CONCLUSIONS The combined use of bioinformatics tools and proteomic profiling provides an effective approach to screen for potential tumor markers. Comparison of plasma profiles from patients with and without known ovarian cancer uncovered a panel of potential biomarkers for detection of ovarian cancer with discriminatory power complementary to that of CA125. Additional studies are required to further validate these biomarkers.

A smartphone dongle for diagnosis of infectious diseases at the point of care

A smartphone accessory can perform a point-of-care test that simultaneously detects three infectious disease markers from fingerprick whole blood in 15 min, as operated by health care workers trained on a software app. Dongle + app = mobile test for sexually transmitted diseases There are thousands of health-related “apps” for smartphones, from tracking sleep patterns to recording heart rate to logging caloric intake. The power of such apps in connecting resource-limited communities to health care workers and, in turn, to proper and immediate care is now emerging. In this issue, Laksanasopin and colleagues describe a microfluidic-based diagnostic test for HIV and syphilis that attaches to (and is powered by) the iPod’s headphone jack. The mobile test also comes complete with an easy-to-use app, flashing test results on-screen in under 15 min. The test is based on the standard immunoassay but uses gold-labeled antibodies to detect HIV and syphilis antigens in only 2 μl of whole blood, and then silver reagents to amplify the resulting signal. The authors deployed the dongle in Rwanda, testing its sensitivity and specificity on 96 patients. Evaluated side by side with the gold standard tests for HIV and syphilis, the dongle produced results with a sensitivity and specificity needed for making treatment decisions in the field. In a survey, a vast majority of patients reported satisfaction with dongle performance. After a few next-generation tweaks, including reducing the size of the dongle, the entire diagnostic package is ready for adoption in resource-poor clinics and communities, to improve detection of HIV and syphilis and empower health care workers to administer timely and appropriate treatments. This work demonstrates that a full laboratory-quality immunoassay can be run on a smartphone accessory. This low-cost dongle replicates all mechanical, optical, and electronic functions of a laboratory-based enzyme-linked immunosorbent assay (ELISA) without requiring any stored energy; all necessary power is drawn from a smartphone. Rwandan health care workers used the dongle to test whole blood obtained via fingerprick from 96 patients enrolling into care at prevention of mother-to-child transmission clinics or voluntary counseling and testing centers. The dongle performed a triplexed immunoassay not currently available in a single test format: HIV antibody, treponemal-specific antibody for syphilis, and nontreponemal antibody for active syphilis infection. In a blinded experiment, health care workers obtained diagnostic results in 15 min from our triplex test that rivaled the gold standard of laboratory-based HIV ELISA and rapid plasma reagin (a screening test for syphilis), with sensitivity of 92 to 100% and specificity of 79 to 100%, consistent with needs of current clinical algorithms. Patient preference for the dongle was 97% compared to laboratory-based tests, with most pointing to the convenience of obtaining quick results with a single fingerprick. This work suggests that coupling microfluidics with recent advances in consumer electronics can make certain laboratory-based diagnostics accessible to almost any population with access to smartphones.

Serum Mesothelin for Diagnosing Malignant Pleural Mesothelioma: An Individual Patient Data Meta-Analysis

PURPOSE Mesothelin is currently considered the best available serum biomarker of malignant pleural mesothelioma. To examine the diagnostic accuracy and use of serum mesothelin in early diagnosis, we performed an individual patient data (IPD) meta-analysis. METHODS The literature search identified 16 diagnostic studies of serum mesothelin, measured with the Mesomark enzyme-linked immunosorbent assay. IPD of 4,491 individuals were collected, including several control groups and 1,026 patients with malignant pleural mesothelioma. Mesothelin levels were standardized for between-study differences and age, after which the diagnostic accuracy and the factors affecting it were examined with receiver operating characteristic (ROC) regression analysis. RESULTS At a common diagnostic threshold of 2.00 nmol/L, the sensitivities and specificities of mesothelin in the different studies ranged widely from 19% to 68% and 88% to 100%, respectively. This heterogeneity can be explained by differences in study population, because type of control group, mesothelioma stage, and histologic subtype significantly affected the diagnostic accuracy. The use of mesothelin in early diagnosis was evaluated by differentiating 217 patients with stage I or II epithelioid and biphasic mesothelioma from 1,612 symptomatic or high-risk controls. The resulting area under the ROC curve was 0.77 (95% CI, 0.73 to 0.81). At 95% specificity, mesothelin displayed a sensitivity of 32% (95% CI, 26% to 40%). CONCLUSION In patients suspected of having mesothelioma, a positive blood test for mesothelin at a high-specificity threshold is a strong incentive to urge further diagnostic steps. However, the poor sensitivity of mesothelin clearly limits its added value to early diagnosis and emphasizes the need for further biomarker research.

Cancer proteomics: Serum diagnostics for tumor marker discovery.

Abstract: Cancer proteomics is an exciting field that is witnessing many new developments in recent years. It is hoped that these advances will result in decreased cancer death rates, which have not declined dramatically in the last several decades. Some of the problems with current tumor markers include the lack of sensitivity and specificity, factors that prevent their use in population‐based screening of disease. Thus, there is an urgent need to identify novel biomarkers that can faithfully detect the disease state. As we are now in the post‐genome era, many opportunities have been created. Genomic sequence data are available for human, as well as several other species. We are now poised to mine these data and to determine the functions of the encoded proteins constituting the human genome. Proteomics affords this opportunity by providing enhanced procedures and tools for discovery and also a framework for understanding these components in terms of pathogenesis. New technologies and improvements in existing methodologies will allow for the rapid growth in the identification and characterization of peptides and proteins that are unique to various clinical states. This technology can be successfully applied to clinical specimens for the identification of new tumor markers.

Novel Serum and Urine Markers for Pediatric Appendicitis

OBJECTIVES The objective was to describe the association between two novel biomarkers, calprotectin and leucine-rich alpha glycoprotein-1 (LRG), and appendicitis in children. METHODS This was a prospective, cross-sectional study of children 3 to 18 years old presenting to a pediatric emergency department (ED) with possible appendicitis. Blood and urine samples were assayed for calprotectin and LRG via enzyme-linked immunosorbent assay (ELISA). Final diagnosis was determined by histopathology or telephone follow-up. Biomarker levels were compared for subjects with and without appendicitis. Recursive partitioning was used to identify thresholds that predicted appendicitis. RESULTS Of 176 subjects, mean (±SD) age was 11.6 (±4.0) years and 52% were male. Fifty-eight patients (34%) were diagnosed with appendicitis. Median plasma calprotectin, serum LRG, and urine LRG levels were higher in appendicitis versus nonappendicitis (p < 0.008). When stratified by perforation status, median plasma calprotectin and serum LRG levels were higher in nonperforated appendicitis versus nonappendicitis (p < 0.01). Median serum LRG, urine LRG, and plasma calprotectin levels were higher in perforated appendicitis compared to nonperforated appendicitis (p < 0.05). Urine calprotectin did not differ among groups. A serum LRG < 40,150 ng/mL, a urine LRG < 42 ng/mL, and a plasma calprotectin < 159 ng/mL, each provided a sensitivity and negative predictive value of 100% to identify children at low risk for appendicitis, but with specificities ranging from 23% to 35%. The standard white blood cell (WBC) count achieved 100% sensitivity at a higher specificity than both novel biomarkers. CONCLUSIONS Plasma calprotectin and serum/urine LRG are elevated in pediatric appendicitis. No individual marker performed as well as the WBC count.

An optimized procedure for exosome isolation and analysis using serum samples: Application to cancer biomarker discovery.

Exosomes are RNA and protein-containing nanovesicles secreted by all cell types and found in abundance in body fluids, including blood, urine and cerebrospinal fluid. These vesicles seem to be a perfect source of biomarkers, as their cargo largely reflects the content of parental cells, and exosomes originating from all organs can be obtained from circulation through minimally invasive or non-invasive means. Here we describe an optimized procedure for exosome isolation and analysis using clinical samples, starting from quick and robust extraction of exosomes with Total exosome isolation reagent, then isolation of RNA followed by qRT-PCR. Effectiveness of this workflow is exemplified by analysis of the miRNA content of exosomes derived from serum samples - obtained from the patients with metastatic prostate cancer, treated prostate cancer patients who have undergone prostatectomy, and control patients without prostate cancer. Three promising exosomal microRNA biomarkers were identified, discriminating these groups: hsa-miR375, hsa-miR21, hsa-miR574.

Artifactual isoform profile modification following treatment of human plasma or serum with protease inhibitor, monitored by 2-dimensional electrophoresis and mass spectrometry.

The inclusion of protease inhibitors in serum or plasma samples has been found to significantly impact the isoform profile of selected plasma proteins as seen on 2-dimensional electrophoresis (2-DE) gels. With the addition of a protease inhibitor cocktail, several human plasma protein trains [depleted of albumin and immunoglobulin G (IgG)] exhibited higher isoelectric point (pI) isoforms. This shift was especially apparent for apolipoprotein A1 (apo A1), a relatively high abundance protein. The six protease inhibitor components of the cocktail were individually investigated with albumin and IgG depleted human plasma, and it was shown that the observed effects were caused by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor that covalently modifies proteins and/or peptides. Several serine-and/or tyrosine-containing peptides of apo A1 were modified with a concomitant mass increase of 183 Da, which is consistent with the mass increase expected following reaction with AEBSF. These modifications were observed with increasing propensity in the higher pI spots. An increase in both the number and proportion of modified peptides with increasing pI was also observed. A model is proposed for the random or stochastic coupling of AEBSF-derived moieties to serine and/or tyrosine residues throughout apo A1 and potentially other plasma proteins.

Circulating tumor DNA analysis in patients with cancer: American society of clinical oncology and college of American pathologists joint review

Purpose Clinical use of analytical tests to assess genomic variants in circulating tumor DNA (ctDNA) is increasing. This joint review from ASCO and the College of American Pathologists summarizes current information about clinical ctDNA assays and provides a framework for future research. Methods An Expert Panel conducted a literature review on the use of ctDNA assays for solid tumors, including pre-analytical variables, analytical validity, interpretation and reporting, and clinical validity and utility. Results The literature search identified 1,338 references. Of those, 390, plus 31 references supplied by the Expert Panel, were selected for full-text review. There were 77 articles selected for inclusion. Conclusion The evidence indicates that testing for ctDNA is optimally performed on plasma collected in cell stabilization or EDTA tubes, with EDTA tubes processed within 6 hours of collection. Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity and clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial. Given the rapid pace of research, re-evaluation of the literature will shortly be required, along with the development of tools and guidance for clinical practice.

Soluble mesothelin related peptides (SMRP) and osteopontin as protein biomarkers for malignant mesothelioma: analytical validation of ELISA based assays and characterization at mRNA and protein levels

Abstract Background: There is a need to identify reliable markers for malignant mesothelioma. Soluble mesothelin related peptides (SMRP) and osteopontin (OPN) have gained interest in recent years for this purpose. Methods: SMRP (Fujirebio Diagnostics Inc.) and OPN (R&D Inc.) ELISA methods were evaluated for multiple parameters. Concentrations were measured in blood from patients with mesothelioma, normal healthy volunteers, and patients with other (non-mesothelioma) cancers. In silico analysis was performed using the GeoProfiles database. At the protein level, SMRP and OPN were measured in cell culture supernatants, and values were compared in patients pre- and post-extrapleural pneumonectomy. Results: The SMRP assay demonstrates intra-assay CVs of 2.3% and 3% (at 4.6 nM and 13.7 nM, respectively), and inter-assay CVs of 3.5% and 3.7% at the same concentrations. The limit of detection (LOD) is 0.182 nM. The OPN assay demonstrates intra-assay CVs of 5.8%, 4.1%, and 5.2% (at 1.9, 5.1, and 11.1 ng/mL, respectively), and inter-assay CVs of 8.5%, 8.4%, and 12.1% at the same concentrations. The LOD is 0.032 ng/mL. Both SMRP and OPN in mesothelioma patients were significantly higher than in patients with other (non-mesothelioma) cancer and in healthy volunteers. The two markers do not appear to correlate with each other and exhibit different tissue expression patterns. Protein concentrations of these markers are highest in different sets of cell lines. Finally, SMRP but not OPN concentrations were decreased in five of seven consecutive patients after extrapleural pneumonectomy, compared to their respective pre-operative values. Conclusions: These assays provide reliable and reproducible quantitation of SMRP and OPN proteins. Both are increased in mesothelioma patients compared to non-mesothelioma controls. However, the two analytes do not correlate with each other and show distinct expression profiles and protein expression. Concentrations of SMRP but not OPN are decreased in post-surgical samples. Our results further characterize these markers, establish assay performance characteristics, and lay the groundwork for further studies to measure these markers in blood. Clin Chem Lab Med 2010;48:271–8.