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1.  Activated Leukocyte Cell Adhesion Molecule: a Novel Biomarker for Breast Cancer 
Activated leukocyte cell adhesion molecule (ALCAM) has been implicated in tumorigenesis. Our goal was to examine the levels of ALCAM, in addition to the classical breast cancer tumor markers carbohydrate antigen 15-3 (CA15-3) and carcinoembryonic antigen (CEA), in serum by quantitative ELISA for diagnosis in breast cancer patients. The three proteins were measured in serum of 100 healthy women, 50 healthy men and 150 breast carcinoma patients. The diagnostic sensitivity and specificity of the tests were calculated and the association of serum marker concentrations with various clinicopathologic variables was examined using nonparametric Kruskal-Wallis tests. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic performance of the biomarkers. ALCAM, with area under the curve (AUC) of 0.78 [95% CI: 0.73, 0.84] outperformed CA15-3 (AUC= 0.70 [95% CI: 0.64, 0.76]) and CEA (AUC= 0.63 [95% CI: 0.56, 0.70]). The incremental values of AUC for ALCAM over that for CA15-3 were statistically significant (Delong test, p <0.05). Combining CA15-3 and ALCAM yielded a ROC curve with an AUC of 0.81 (95% CI [0.75, 0.87]). Serum ALCAM appears to be a new biomarker for breast cancer and may have value for disease diagnosis.
doi:10.1002/ijc.24292
PMCID: PMC3743675  PMID: 19322904
ALCAM; breast cancer; immunoassay; immunoglobulin superfamily; tumor marker
2.  Proteomic Signatures of the Desmoplastic Invasion Front Reveal Collagen Type XII as a Marker of Myofibroblastic Differentiation During Colorectal Cancer Metastasis 
Oncotarget  2012;3(3):267-285.
Cancer-associated fibroblasts (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by growth factors secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident fibroblasts during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a 22-protein “myofibroblastic signature” with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myofibroblasts, and/or cancer cells undergoing dedifferentiation.
PMCID: PMC3359884  PMID: 22408128
Colorectal cancer; Cancer-associated fibroblasts; Desmoplasia; Proteomics; Secretome; Collagen type XII
3.  Genomic Instability and Copy-Number Heterogeneity of Chromosome 19q, Including the Kallikrein Locus, in Ovarian Carcinomas 
Molecular oncology  2010;5(1):48-60.
Many tissue kallikrein (KLK) genes and proteins are candidate diagnostic, prognostic and predictive biomarkers for ovarian cancer (OCa). We previously demonstrated that the KLK locus (19q13.3/4) is subject to copy-number gains and structural rearrangements in a pilot study of cell lines and ovarian cancer primary tissues, shown to overexpress KLK gene family members. To determine the overall frequency of genomic instability and copy-number changes, a retrospective study was conducted using formalin-fixed paraffin embedded (FFPE) tissues. Eighty-one chemotherapy naïve serous OCas were examined using 3-colour fluorescence in situ hybridization (FISH) to identify structural and numerical changes on 19q, including the KLK locus; in addition to immunohistochemistry (IHC) for KLK6, which has been shown to be overexpressed in OCa. The KLK locus was subject to copy-number changes in ~83% of cases: net gain in 51%, net loss in 30% and amplified in 2%; and found to be chromosomally unstable (p<0.001). All cases showed a wide range of immuoreactivity for KLK6 by IHC. Although no strong correlation could be found with copy number, the latter was contributing factor to the observed KLK6 protein overexpression. Moreover, univariate and multivariate analyses showed an association between the net loss of the KLK locus with longer disease-free survival. Interestingly, FISH analyses indicated that chromosome 19q was subject to structural rearrangement in 62% of cases and was significantly correlated to tumor grade (p<0.001). We conclude that numerical and structural aberrations of chromosome 19q, affect genes including the KLK gene members, may contributing to ovarian carcinoma progression and aggressiveness.
doi:10.1016/j.molonc.2010.08.002
PMCID: PMC3110681  PMID: 20800559
FISH; copy number; chromosomal instability; structural rearrangement; kallikrein locus; ovarian carcinoma
4.  Cancer Biomarkers: Can We Turn Recent Failures into Success? 
Disease biomarkers are used widely in medicine. But very few biomarkers are useful for cancer diagnosis and monitoring. Over the past 15 years, major investments have been made to discover and validate cancer biomarkers. Despite such investments, no new major cancer biomarkers have been approved for clinical use for at least 25 years. In the last decade, many reports have described new cancer biomarkers that promised to revolutionize the diagnosis of cancer and the management of cancer patients. However, many initially promising biomarkers have not been validated for clinical use. In this commentary, a plethora of parameters before sample analysis, during sample analysis, and after sample analysis that can complicate biomarker discovery and validation and lead to “false discovery” are discussed. Several examples of biomarker discoveries that were published in high-profile journals are also presented, as well as why they were not validated and the lessons learned from these false discoveries, so that similar mistakes can be avoided in the future.
doi:10.1093/jnci/djq306
PMCID: PMC2950166  PMID: 20705936
5.  Nidogen-2: A New Serum Biomarker for Ovarian Cancer 
Clinical biochemistry  2009;43(4-5):355-361.
Objectives
New ovarian cancer biomarkers suitable for early disease diagnosis, prognosis or monitoring could improve patient management and outcomes.
Design and Methods
Nidogen-2 was measured by immunoassay in serum of 100 healthy women, 100 women with benign gynecological conditions and 100 women with ovarian carcinoma.
Results
Serum nidogen-2 concentration between normal and benign disease patients was not different (median, 13.2 and 12.1 μg/L, respectively). However, nidogen-2 concentration in serum of ovarian cancer patients was elevated (median, 18.6 μg/L; p<0.0001). Both nidogen-2 and CA125 were elevated more in serous histotypes of ovarian cancer and late state disease. Nidogen-2 and CA125 concentrations were strongly correlated. ROC curve analysis for nidogen-2 had an area under the curve (AUC) ranging from 0.73 to 0.83 but CA125 was superior (AUC ranging from 0.87 to 0.99). There was no complementarity between the two markers.
Conclusions
Nidogen-2 is a new biomarker for ovarian cancer which correlates closely with CA125.
doi:10.1016/j.clinbiochem.2009.10.012
PMCID: PMC3109863  PMID: 19883638
Ovarian cancer; biomarker; proteomics; mass spectrometry; nidogen-2; basement membrane
6.  Quantitative tandem mass-spectrometry of skin tissue reveals putative psoriatic arthritis biomarkers 
Clinical Proteomics  2015;12(1):1.
Background
Psoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA.
Methods
Skin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins.
Results
Forty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001).
Conclusion
Proteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology.
Electronic supplementary material
The online version of this article (doi:10.1186/1559-0275-12-1) contains supplementary material, which is available to authorized users.
doi:10.1186/1559-0275-12-1
PMCID: PMC4304122
Psoriatic arthritis; Cutaneous psoriasis; Proteomics; Mass spectrometry; Biomarker
7.  The hundred person wellness project and Google’s baseline study: medical revolution or unnecessary and potentially harmful over-testing? 
BMC Medicine  2015;13:5.
The Hundred Person Wellness Project is an ambitious pilot undertaking, which aims to intensely monitor 100 individuals over 10 months. Patients with abnormal findings will be treated, in hopes that this early intervention will avoid, or delay, symptomatic disease. Google’s “Baseline Study” is of similar scope and will enroll 10,000 people over 2 to 3 years. I here speculate that these approaches will likely not be effective in preventing disease, but instead, lead to unnecessary and potentially harmful interventions. Examples from the cancer screening experience over the last 30 years are provided, which show that intensive testing may uncover indolent disease or incidental findings which, when treated, may cause more harm than good. Additional examples show that aggressive treatments for cancer and other diseases do not always lead to better patient outcomes. I conclude that the recent advances in omics provide us with unprecedented opportunities for high content clinical testing, but such testing should be used with caution to avoid the harmful consequences of over-diagnosis and over-treatment. Despite the detailed rebuttals by Hood and colleagues in another commentary in BMC Medicine, time will show the actual benefits and harms of these ambitious initiatives.
Please see related commentary: http://dx.doi.org/10.1186/s12916-014-0238-7
doi:10.1186/s12916-014-0239-6
PMCID: PMC4288557  PMID: 25575898
Cancer screening; Incidental findings; Indolent disease; Over-diagnosis; Over-treatment; Population screening
8.  Kallikrein Cascades in Traumatic Spinal Cord Injury: In Vitro Evidence for Roles in Axonopathy and Neuron Degeneration 
Kallikreins (KLKs) are a family of 15 secreted serine proteases with emerging roles in neurological disease. To illuminate their contributions to the pathophysiology of spinal cord injury (SCI) we evaluated acute through chronic changes in the immunohistochemical appearance of six kallikreins, KLK1, KLK5, KLK6, KLK7, KLK8 and KLK9 in post-mortem human traumatic SCI cases, quantified their RNA expression levels in experimental murine SCI, and assessed the impact of recombinant forms of each enzyme toward murine cortical neurons in vitro. Temporally and spatially distinct changes in kallikrein expression were observed with partially overlapping patterns between human and murine SCI, including peak elevations (or reductions) during the acute and subacute periods. KLK9 showed the most robust changes and remained elevated chronically. Importantly, a subset of kallikreins, KLK1, KLK5, KLK6, KLK7 and KLK9 were shown to be neurotoxic toward primary neurons in vitro. Kallikrein immunoreactivity was also observed in association with swollen axons and retraction bulbs in the human SCI materials examined. Together, these findings demonstrate that elevated levels of a significant subset of kallikreins are positioned to contribute to neurodegenerative changes in cases of CNS trauma and disease and therefore represent new targets for the development of neuroprotective strategies.
doi:10.1097/NEN.0000000000000007
PMCID: PMC4097185  PMID: 24128681
axonal injury; degeneration; inflammation; neural injury; spinal cord injury
9.  OVSCORE - a validated score to identify ovarian cancer patients not suitable for primary surgery 
Oncology Letters  2014;9(1):418-424.
Following primary debulking surgery, the presence of a residual tumor mass is one of the most important prognostic factors in ovarian cancer. In a previous study, we established the OVSCORE, an algorithm to predict surgical outcome, based on the clinical factors of nuclear grading and ascitic fluid volume, plus the cancer biomarkers, kallikrein-related peptidases (KLKs), KLK6 and KLK13. In the present study, OVSCORE performance was tested in an independent ovarian cancer patient cohort consisting of 87 patients. The impact of KLKs, KLK5, 6, 7 and 13 and other clinical factors on patient prognosis and outcome was also evaluated. The OVSCORE proved to be a strong and statistically significant predictor of surgical success in terms of area under the receiver operating characteristic curve (ROC AUC, 0.777), as well as positive and negative predictive value in this independent study group. KLK6 and 13 individually did not show clinical relevance in this cohort, but two other KLKs, KLK5 and KLK7, were associated with advanced FIGO stage, higher nuclear grade and positive lymph node status. In the multivariate Cox regression analysis for overall survival (OS), KLK7 had a protective impact on OS. This study confirms the role of KLKs in ovarian cancer for surgical success and survival, and validates the novel OVSCORE algorithm in an independent collective. As a key clinical application, the OVSCORE could aid gynecological oncologists in identifying those ovarian cancer patients unlikely to benefit from radical surgery who could be candidates for alternative therapeutic approaches.
doi:10.3892/ol.2014.2630
PMCID: PMC4246645  PMID: 25436002
kallikrein-related peptidases; kallikrein; ovarian cancer; surgical success; residual tumor; prognosis
10.  Performance Evaluation of Siemens ADVIA Centaur and Roche MODULAR Analytics E170 Total 25-OH Vitamin D Assays 
Clinical biochemistry  2012;45(0):1485-1490.
Objectives
To evaluate the newly developed Roche MODULAR Analytics E170 Total Vitamin D and the Siemens ADVIA Centaur® Vitamin D Total assays.
Materials and Methods
Assays were evaluated using the Clinical and Laboratory Standards Institute protocols. Split patient samples were compared with LC-MS/MS and DiaSorin LIAISON assays (n=79 including 15 specimens with detectable endogenous 25-OH vitamin D2). Assay accuracy was also evaluated using the Vitamin D External Quality Assessment Scheme samples.
Results
The ADVIA Centaur and E170 assays demonstrated maximum total CVs of 14.1% and 5.9%, respectively. Both showed excellent linearity (R2 >0.99). The ADVIA Centaur assay demonstrated interference with bilirubin at 800 μmol/L, hemolysis at 1.25 g/L, and triglycerides at 2.8 mmol/L. Compared to LC-MS/MS, the ADVIA Centaur assay demonstrated a R2 value of 0.893, average bias of −8.8%; the E170 assay an R2 value of 0.872, average bias of 14.3% with underestimation of 25-OH vitamin D2. Compared to the LIAISON assay, the ADVIA Centaur assay demonstrated an R2 value of 0.781, average bias of −17.3%; the E170 assay an R2 value of 0.823, average bias of 11.4%. The ADVIA Centaur and E170 assays demonstrated a biases of <20% in 10/10 and 8/10 samples, respectively.
Conclusions
The ADVIA Centaur and E170 vitamin D assays demonstrated acceptable linearity, imprecision, and accuracy. The E170 assay demonstrated consistent underestimation of 25-OH vitamin D2 levels. Compared with LC-MS/MS, the ADVIA Centaur assay demonstrated a higher R2 value and a smaller average bias than the E170 assay.
doi:10.1016/j.clinbiochem.2012.06.002
PMCID: PMC4193681  PMID: 22705028
25-OH vitamin D; immunoassay; method comparison; LC-MS/MS
11.  Induction of complement C3a receptor responses by kallikrein-related peptidase 14 
Activation of the complement system is primarily initiated by pathogen- and damage-associated molecular surface patterns on cellular surfaces. However, there is increasing evidence for direct activation of individual complement components by extrinsic proteinases as part of an intricate crosstalk between physiological effector systems. We hypothesized that kallikrein-related peptidases (KLKs), previously known to regulate inflammation via proteinase-activated receptors, can also play a substantial role in innate immune responses via complement. Indeed, KLKs exemplified by KLK14 were efficiently able to cleave C3, the point of convergence of the complement cascade, indicating a potential modulation of C3-mediated functions. By using in vitro fragmentation assays, mass spectrometric analysis, and cell signaling responses, we pinpointed the generation of the C3a fragment of C3 as a product with potential biological activity released by the proteolytic action of KLK14. Using mice with various complement deficiencies, we demonstrated that the intraplantar administration of KLK14 results in C3-associated paw edema. The edema response was dependent on the presence of the receptor for C3a but was not associated with the receptor for the downstream complement effector C5a. Our findings point to C3 as one of the potential substrates of KLKs during inflammation. Given the wide distribution of the KLKs in tissues and biological fluids where complement components may also be expressed, we suggest that via C3 processing, tissue-localized KLKs can play an extrinsic complement-related role during activation of the innate immune response.
doi:10.4049/jimmunol.1202999
PMCID: PMC3922206  PMID: 24014879
anaphylatoxin; C3a receptor; complement; inflammation; kallikrein-related peptidases; paw edema; trypsin
12.  Towards identification of true cancer biomarkers 
BMC Medicine  2014;12(1):156.
Background
Most of newly discovered cancer biomarkers fail in the clinic because they lack sensitivity and/or specificity. The current explosion in knowledge of the mutational spectrum of many cancer types, as a result of whole exome and whole genome sequencing, has revealed a wide spectrum of mutations that appear to be highly specific for various cancer types.
Discussion
Mass spectrometry (MS) has the ability to monitor tryptic peptides in complex biological mixtures with high sensitivity and specificity. It may be possible in the near future to combine the known spectrum of gene mutations revealed by genomics with the power of MS, in order to quantify mutant peptides that are highly specific for cancer, in a multiplex fashion. Such mutant peptides, quantified in the circulation and other fluids, may represent tumor markers that are suitable for detection and monitoring of cancer.
Summary
The power of genomic and proteomic technologies can be combined to identify highly specific analytes for biomarker applications.
doi:10.1186/s12916-014-0156-8
PMCID: PMC4161831  PMID: 25220599
Cancer biomarker; Genomics; Proteomics; Mutant proteins; High specificity; Selected reaction monitoring; Mass spectrometry
13.  Q & A # 13 Pheochromocytoma 
Clinical chemistry  2012;59(3):466-472.
doi:10.1373/clinchem.2012.182246
PMCID: PMC4139095  PMID: 23209036
14.  Identification of psoriatic arthritis mediators in synovial fluid by quantitative mass spectrometry 
Clinical Proteomics  2014;11(1):27.
Background
Synovial fluid (SF) is a dynamic reservoir for proteins originating from the synovial membrane, cartilage, and plasma, and may therefore reflect the pathophysiological conditions that give rise to arthritis. Our goal was to identify and quantify protein mediators of psoriatic arthritis (PsA) in SF.
Methods
Age and gender-matched pooled SF samples from 10 PsA and 10 controls [early osteoarthritis (OA)], were subjected to label-free quantitative proteomics using liquid chromatography coupled to mass spectrometry (LC-MS/MS), to identify differentially expressed proteins based on the ratios of the extracted ion current of each protein between the two groups. Pathway analysis and public database searches were conducted to ensure these proteins held relevance to PsA. Multiplexed selected reaction monitoring (SRM) assays were then utilized to confirm the elevated proteins in the discovery samples and in an independent set of samples from patients with PsA and controls.
Results
We determined that 137 proteins were differentially expressed between PsA and control SF, and 44 were upregulated. The pathways associated with these proteins were acute-phase response signalling, granulocyte adhesion and diapedesis, and production of nitric oxide and reactive oxygen species in macrophages. The expression of 12 proteins was subsequently quantified using SRM assays.
Conclusions
Our in-depth proteomic analysis of the PSA SF proteome identified 12 proteins which were significantly elevated in PsA SF compared to early OA SF. These proteins may be linked to the pathogenesis of PsA, as well serve as putative biomarkers and/or therapeutic targets for this disease.
doi:10.1186/1559-0275-11-27
PMCID: PMC4108225  PMID: 25097465
Psoriatic arthritis; Early osteoarthritis; Proteomics; Mass spectrometry; Synovial fluid; Selected reaction monitoring assays; Proteins; Mediators
15.  Deciphering the peptidome of urine from ovarian cancer patients and healthy controls 
Clinical proteomics  2014;11(1):23.
Background
Ovarian cancer (OvCa) is the most lethal gynecological malignancy. The emergence of high-throughput technologies, such as mass spectrometry, has allowed for a paradigm shift in the way we search for novel biomarkers. Urine-based peptidomic profiling is a novel approach that may result in the discovery of noninvasive biomarkers for diagnosing patients with OvCa. In this study, the peptidome of urine from 6 ovarian cancer patients and 6 healthy controls was deciphered.
Results
Urine samples underwent ultrafiltration and the filtrate was subjected to solid phase extraction, followed by fractionation using strong cation exchange chromatography. These fractions were analyzed using an Orbitrap mass spectrometer. Over 4600 unique endogenous urine peptides arising from 713 proteins were catalogued, representing the largest urine peptidome reported to date. Each specimen was processed in triplicate and reproducibility at the protein (69-76%) and peptide (58-63%) levels were noted. More importantly, over 3100 unique peptides were detected solely in OvCa specimens. One such promising biomarker was leucine-rich alpha-2-glycoprotein (LRG1), where multiple peptides were found in all urines from OvCa patients, but only one peptide was found in one healthy control urine sample.
Conclusions
Mining the urine peptidome may yield highly promising novel OvCa biomarkers.
doi:10.1186/1559-0275-11-23
PMCID: PMC4065538  PMID: 24982608
Biomarker; Early diagnosis; Mass spectrometry; Ovarian cancer; Urine peptidome
17.  Strategies for discovering novel pancreatic cancer biomarkers 
Journal of proteomics  2012;81:126-134.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in both men and women in Canada and the United States and has the most dismal survival rates among any solid malignancy. Most patients are diagnosed with pancreatic cancer once the disease has progressed into an advanced or metastatic stage, making the only curative approach of resection surgery impossible. The persistent delayed or missed diagnosis of pancreatic cancer can be attributed to the absence of early symptoms and the lack of efficient non-invasive screening or diagnostic tests in clinical practice. Given that earlier diagnosis is critical for ameliorating patients’ survival rates, there is an urgent need for biomarkers with enough sensitivity and specificity to help diagnose pancreatic cancer early. Serological biomarkers provide a minimally invasive and efficient way of detecting pancreatic cancer, however, there is currently no marker with sufficient diagnostic sensitivity and specificity to identify early cancer patients. This review focuses on the classical tumour markers for PDAC as well as emerging markers. In addition, we will discuss an integrative proteomic approach used in our lab to identify a panel of biomarkers that have the potential to allow the early detection of PDAC.
doi:10.1016/j.jprot.2012.09.025
PMCID: PMC3561491  PMID: 23026552
18.  Deciphering the ovarian cancer ascites fluid peptidome 
Clinical Proteomics  2014;11(1):13.
Background
Conventional proteomic approaches have thus far been unable to identify novel serum biomarkers for ovarian cancer that are more sensitive and specific than the current clinically used marker, CA-125. Because endogenous peptides are smaller and may enter the circulation more easily than proteins, a focus on the low-molecular-weight region may reveal novel biomarkers with enhanced sensitivity and specificity. In this study, we deciphered the peptidome of ascites fluid from 3 ovarian cancer patients and 3 benign individuals (ascites fluid from patients with liver cirrhosis).
Results
Following ultrafiltration of the ascites fluids to remove larger proteins, each filtrate was subjected to solid phase extraction and fractionated using strong cation exchange chromatography. The resultant fractions were analyzed using an Orbitrap mass spectrometer. We identified over 2000 unique endogenous peptides derived from 259 proteins. We then catalogued over 777 peptides that were found only in ovarian cancer ascites. Our list of peptides found in ovarian cancer specimens includes fragments derived from the proteins vitronectin, transketolase and haptoglobin.
Conclusions
Peptidomics may uncover previously undiscovered disease-specific endogenous peptides that warrant further investigation as biomarkers for ovarian cancer.
doi:10.1186/1559-0275-11-13
PMCID: PMC4230032  PMID: 24694173
Biomarker; Early diagnosis; Mass spectrometry; Ovarian cancer; Ascites fluid; Peptidome
19.  Emerging clinical importance of the cancer biomarkers kallikrein-related peptidases (KLK) in female and male reproductive organ malignancies 
Radiology and Oncology  2013;47(4):319-329.
Background
Tumor tissue-associated KLKs (kallikrein-related peptidases) are clinically important biomarkers that may allow prognosis of the cancer disease and/or prediction of response/failure of cancer patients to cancer-directed drugs. Regarding the female/male reproductive tract, remarkably, all of the fifteen KLKs are expressed in the normal prostate, breast, cervix uteri, and the testis, whereas the uterus/endometrium and the ovary are expressing a limited number of KLKs only.
Conclusions
Most of the information regarding elevated expression of KLKs in tumor-affected organs is available for ovarian cancer; depicting them as valuable biomarkers in the cancerous phenotype. In contrast, for breast cancer, a series of KLKs was found to be downregulated. However, in breast cancer, KLK4 is elevated which is also true for ovarian and prostate cancer. In such cases, selective synthetic KLK inhibitors that aim at blocking the proteolytic activities of certain KLKs may serve as future candidate therapeutic drugs to interfere with tumor progression and metastasis.
doi:10.2478/raon-2013-0053
PMCID: PMC3814276  PMID: 24294176
cancer, proteases; endometrium; ovary; uterus; prostate; testis; cervix; breast
20.  Targeted Selected Reaction Monitoring Mass Spectrometric Immunoassay for Insulin-like Growth Factor 1 
PLoS ONE  2013;8(11):e81125.
Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins. The workflow also includes an immunoaffinity step using antibody-derivatized pipette tips, followed by elution, trypsin digestion, and LC-MS/MS separation and detection of the signature peptides in a selected reaction monitoring (SRM) mode. The resulting quantitative mass spectrometric immunoassay (MSIA) exhibited good linearity in the range of 1 to 1,500 ng/mL IGF1, intra- and inter-assay precision with CVs of less than 10%, and lowest limits of detection of 1 ng/mL. The linearity and recovery characteristics of the assay were also established, and the new method compared to a commercially available immunoassay using a large cohort of human serum samples. The IGF1 SRM MSIA is well suited for use in clinical laboratories.
doi:10.1371/journal.pone.0081125
PMCID: PMC3836743  PMID: 24278387
21.  Interlaboratory Reproducibility of Selective Reaction Monitoring Assays Using Multiple Upfront Analyte Enrichment Strategies 
Journal of proteome research  2012;11(8):3986-3995.
Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays. Furthermore, an important caveat of SRM-based assays for proteins is that many low-abundance analytes require some type of enrichment before detection with MS. This adds a level of complexity to the procedure and the potential for irreproducibility increases, especially across different laboratories with different operators. The purpose of this study was to test the interlaboratory reproducibility of SRM assays with various upfront enrichment strategies and different types of clinical samples (representing real-world body fluids commonly encountered in routine clinical laboratories). Three different, previously published enrichment strategies for low-abundance analytes and a no-enrichment strategy for high-abundance analytes were tested across four different laboratories using different liquid chromatography-SRM (LC-SRM) platforms and previously developed SOPs. The results demonstrated that these assays were indeed reproducible with coefficients of variation of less than 30% for the measurement of important clinical proteins across all four laboratories in real world samples.
doi:10.1021/pr300014s
PMCID: PMC3761372  PMID: 22639787
SRM assay; mass spectrometry; proteomics; clinical assay
22.  Validation of four candidate pancreatic cancer serological biomarkers that improve the performance of CA19.9 
BMC Cancer  2013;13:404.
Background
The identification of new serum biomarkers with high sensitivity and specificity is an important priority in pancreatic cancer research. Through an extensive proteomics analysis of pancreatic cancer cell lines and pancreatic juice, we previously generated a list of candidate pancreatic cancer biomarkers. The present study details further validation of four of our previously identified candidates: regenerating islet-derived 1 beta (REG1B), syncollin (SYCN), anterior gradient homolog 2 protein (AGR2), and lysyl oxidase-like 2 (LOXL2).
Methods
The candidate biomarkers were validated using enzyme-linked immunosorbent assays in two sample sets of serum/plasma comprising a total of 432 samples (Sample Set A: pancreatic ductal adenocarcinoma (PDAC, n = 100), healthy (n = 92); Sample Set B: PDAC (n = 82), benign (n = 41), disease-free (n = 47), other cancers (n = 70)). Biomarker performance in distinguishing PDAC from each control group was assessed individually in the two sample sets. Subsequently, multiparametric modeling was applied to assess the ability of all possible two and three marker panels in distinguishing PDAC from disease-free controls. The models were generated using sample set B, and then validated in Sample Set A.
Results
Individually, all markers were significantly elevated in PDAC compared to healthy controls in at least one sample set (p ≤ 0.01). SYCN, REG1B and AGR2 were also significantly elevated in PDAC compared to benign controls (p ≤ 0.01), and AGR2 was significantly elevated in PDAC compared to other cancers (p < 0.01). CA19.9 was also assessed. Individually, CA19.9 showed the greatest area under the curve (AUC) in receiver operating characteristic (ROC) analysis when compared to the tested candidates; however when analyzed in combination, three panels (CA19.9 + REG1B (AUC of 0.88), CA19.9 + SYCN + REG1B (AUC of 0.87) and CA19.9 + AGR2 + REG1B (AUC of 0.87)) showed an AUC that was significantly greater (p < 0.05) than that of CA19.9 alone (AUC of 0.82). In a comparison of early-stage (Stage I-II) PDAC to disease free controls, the combination of SYCN + REG1B + CA19.9 showed the greatest AUC in both sample sets, (AUC of 0.87 and 0.92 in Sets A and B, respectively).
Conclusions
Additional serum biomarkers, particularly SYCN and REG1B, when combined with CA19.9, show promise as improved diagnostic indicators of pancreatic cancer, which therefore warrants further validation.
doi:10.1186/1471-2407-13-404
PMCID: PMC3847832  PMID: 24007603
Pancreatic cancer; Serum biomarkers; Biomarker validation; ELISA; Biomarker panel
23.  Quantitative DNA methylation analysis of genes coding for kallikrein-related peptidases 6 and 10 as biomarkers for prostate cancer 
Epigenetics  2012;7(9):1037-1045.
DNA methylation plays an important role in carcinogenesis and is being recognized as a promising diagnostic and prognostic biomarker for a variety of malignancies including Prostate cancer (PCa). The human kallikrein-related peptidases (KLKs) have emerged as an important family of cancer biomarkers, with KLK3, encoding for Prostate Specific Antigen, being most recognized. However, few studies have examined the epigenetic regulation of KLKs and its implications to PCa. To assess the biological effect of DNA methylation on KLK6 and KLK10 expression, we treated PC3 and 22RV1 PCa cells with a demethylating drug, 5-aza-2′deoxycytidine, and observed increased expression of both KLKs, establishing that DNA methylation plays a role in regulating gene expression. Subsequently, we have quantified KLK6 and KLK10 DNA methylation levels in two independent cohorts of PCa patients operated by radical prostatectomy between 2007–2011 (Cohort I, n = 150) and 1998–2001 (Cohort II, n = 124). In Cohort I, DNA methylation levels of both KLKs were significantly higher in cancerous tissue vs. normal. Further, we evaluated the relationship between DNA methylation and clinicopathological parameters. KLK6 DNA methylation was significantly associated with pathological stage only in Cohort I while KLK10 DNA methylation was significantly associated with pathological stage in both cohorts. In Cohort II, low KLK10 DNA methylation was associated with biochemical recurrence in univariate and multivariate analyses. A similar trend for KLK6 DNA methylation was observed. The results suggest that KLK6 and KLK10 DNA methylation distinguishes organ confined from locally invasive PCa and may have prognostic value.
doi:10.4161/epi.21524
PMCID: PMC3515013  PMID: 22874102
biomarkers; epigenetics; kallikrein-related peptidases; prostate cancer; quantitative DNA methylation analysis
24.  Semiquantitative proteomic analysis of human hippocampal tissues from Alzheimer’s disease and age-matched control brains 
Clinical proteomics  2013;10(1):5.
Background
Alzheimer’s disease (AD) is the most common type of dementia affecting people over 65 years of age. The hallmarks of AD are the extracellular deposits known as amyloid β plaques and the intracellular neurofibrillary tangles, both of which are the principal players involved in synaptic loss and neuronal cell death. Tau protein and Aβ fragment 1–42 have been investigated so far in cerebrospinal fluid as a potential AD biomarkers. However, an urgent need to identify novel biomarkers which will capture disease in the early stages and with better specificity remains. High-throughput proteomic and pathway analysis of hippocampal tissue provides a valuable source of disease-related proteins and biomarker candidates, since it represents one of the earliest affected brain regions in AD.
Results
In this study 2954 proteins were identified (with at least 2 peptides for 1203 proteins) from both control and AD brain tissues. Overall, 204 proteins were exclusively detected in AD and 600 proteins in control samples. Comparing AD and control exclusive proteins with cerebrospinal fluid (CSF) literature-based proteome, 40 out of 204 AD related proteins and 106 out of 600 control related proteins were also present in CSF. As most of these proteins were extracellular/secretory origin, we consider them as a potential source of candidate biomarkers that need to be further studied and verified in CSF samples.
Conclusions
Our semiquantitative proteomic analysis provides one of the largest human hippocampal proteome databases. The lists of AD and control related proteins represent a panel of proteins potentially involved in AD pathogenesis and could also serve as prospective AD diagnostic biomarkers.
doi:10.1186/1559-0275-10-5
PMCID: PMC3648498  PMID: 23635041
Alzheimer’s disease; Cerebrospinal fluid; Hippocampus; Human brain; Mass spectrometry
25.  Fascin-1 is a novel biomarker of aggressiveness in some carcinomas 
BMC Medicine  2013;11:53.
Tremendous progress has been made in recent years towards the understanding, prevention and management of malignant disease, yet cancer remains a leading cause of global mortality and morbidity. Current approaches towards combating this disease include prevention, early detection and various treatment modalities. However, even with implementation of novel therapeutic options and preventative measures, most cancers are currently diagnosed at late stages, when treatment therapies are least effective. In a recent study published in BMC Medicine, Tan et al. performed a systematic review and meta-analysis to show that fascin-1, an actin-bundling protein, is associated with increased risk of mortality and metastasis in various cancer types. Although the study examined the association of fascin-1 with mortality, time-to-disease progression, lymph node metastasis and distant metastasis in five major cancer types, the clinical implications of these findings are still unclear and many unanswered questions remain.
Please see related research article here http://www.biomedcentral.com/1741-7015/11/52
doi:10.1186/1741-7015-11-53
PMCID: PMC3626678  PMID: 23443037
diagnosis; fascin-1; immunohistochemistry; prognosis; tumor markers

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