Marker For Diagnosis Of Prostate Cancer Research Articles

Integrative Analysis of Androgen Receptor Interactors Aberrations and Associated Prognostic Significance in Prostate Cancer.

Much progress has been made by directing against the adrogen receptor (AR) pathway in the treatment of prostate cancer in past decades. However, AR-interactors related metastatic castration resistant prostate cancer eventually developed. Here, we aimed to characterize the aberrations and therapeutic implication in advanced disease. STRING database, UALCAN web portal and cBioPortal platform was used to analyze the AR interaction network, gene alterations, as well as the prognostic significance. GO and KEEG analysis was performed to characterize the functional enrichment of the identified AR-interactors. Ten first shell AR-interactors were identified, among of which FOXA1 and PELP1 was significantly up-regulated, while CCND1, CTNNB1, NCOA4 and HSP90AA1 exhibited a significantly decreased pattern. The median survival period of altered group (n = 227) was 70 months (95% CI, 60-105M), while that of non-altered group (n = 545) was 141 months (95% CI, 115.13-NA, P < 0.001). GO and KEGG enrichment showed that the identified AR-interactors were particularly enriched in prostate cancer and thyroid hormone signaling pathway, as well as endocrine resistance. The AR-interactors might be useful markers for prostate cancer diagnosis and prognosis, and provide a new sight for revealing the molecular mechanism of CRPC progression.

The IGSF1, Wnt5a, FGF14, and ITPR1 Gene Expression and Prognosis Hallmark of Prostate Cancer.

Prostate cancer is considered as the second leading cause of cancer related death in men worldwide and the third frequent cancer among Iranian men. Despite the use of PSA as the only biomarker for early diagnosis of prostate cancer, its application in clinical settings is under debate. Therefore, the introduction of new molecular markers for early detection of prostate cancer is needed. In the present study we intended to evaluate the expression of IGSF1, Wnt5a, FGF14, and ITPR1 in prostate cancer specimens by real time PCR. Biopsy samples of 40 prostate cancer cases and 41 healthy Iranian men were compared to determine the relative gene expression of IGSF1, Wnt5a, FGF14, and ITPR1 by real time PCR. Our results showed that Wnt5a, FGF14, and IGSF1 were significantly overexpressed in the prostate cancer patients while the mean relative expression of ITPR1 showed a significant decrease in PCa samples compared to healthy controls. According to results of the present study, the combination panel of IGSF1, Wnt5a, FGF14, and ITPR1 genes could be considered as potential genetic markers for prostate cancer diagnosis. However further studies on larger populations and investigating the clinicopathological relevance of these genes is needed.

STAT3-regulated LncRNA LINC00160 mediates cell proliferation and cell metabolism of prostate cancer cells by repressing RCAN1 expression.

Long non-coding RNA (LncRNA) LINC00160 was reported to be associated with cancer progression and mediates drug resistance. However, the role of LINC00160 in prostate cancer remains unclear. The study sought to study the function of LINC00160 in prostate cancer. Moreover, the potential mechanism was investigated. Silence of LINC00160 inhibited proliferation and promoted the apoptosis of prostate cancer cells, retarded the glycolysis of prostate cancer cells. By acting as a transcription activator, STAT3 induced LINC00160 expression, which regulated RCAN1 transcription epigenetically via binding to EZH2. Mechanically, LINC00160 mediated prostate cell proliferation and metabolism by repressing RCAN1 expression. In summary, LINC00160 may function as the novel marker for prostate cancer diagnosis and therapy.

Blood-Derived Biomarkers of Diagnosis, Prognosis and Therapy Response in Prostate Cancer Patients.

Prostate cancer is among the most frequent cancers in men worldwide. Despite the fact that multiple therapeutic alternatives are available for its treatment, it is often discovered in an advanced stage as a metastatic disease. Prostate cancer screening is based on physical examination of prostate size and prostate-specific antigen (PSA) level in the blood as well as biopsy in suspect cases. However, these markers often fail to correctly identify the presence of cancer, or their positivity might lead to overdiagnosis and consequent overtreatment of an otherwise silent non-progressing disease. Moreover, these markers have very limited if any predictive value regarding therapy response or individual risk for therapy-related toxicities. Therefore, novel, optimally liquid biopsy-based (blood-derived) markers or marker panels are needed, which have better prognostic and predictive value than the ones currently used in the everyday routine. In this review the role of circulating tumour cells, extracellular vesicles and their microRNA content, as well as cellular and soluble immunological and inflammation- related blood markers for prostate cancer diagnosis, prognosis and prediction of therapy response is discussed. A special emphasis is placed on markers predicting response to radiotherapy and radiotherapy-related late side effects.

The Investigation of New Biological Markers for Prostate Cancer Diagnosis and Management in Chinese Population

The Investigation of New Biological Markers for Prostate Cancer Diagnosis and Management in Chinese Population

New Prostate Cancer Targets for Diagnosis, Imaging, and Therapy: Focus on Prostate-Specific Membrane Antigen.

The rising incidence rate of the cancer in the prostate gland has increased the demand for improved diagnostic, imaging, and therapeutic approaches. Prostate-specific membrane antigen (PSMA), with folate hydrolase and carboxypeptidase and, internalization activities, is highly expressed in the epithelial cells of the prostate gland and is strongly upregulated in prostatic adenocarcinoma, with elevated expression correlating with, metastasis, progression, and androgen independence. Recently, PSMA has been an active target of investigation by several approaches, including the successful utilization of small molecule inhibitors, RNA aptamer conjugates, PSMA-based immunotherapy, and PSMA-targeted prodrug therapy. Future investigations of PSMA in prostate cancer (PCa) should focus in particular on its intracellular activities and functions. The objective of this contribution is to review the current role of PSMA as a marker for PCa diagnosis, imaging, and therapy.

Abstract 1476: Cell-free miR-141 as a molecular marker for prostate cancer metastasis

Abstract Prostate cancer is the second most commonly diagnosed neoplasia in men. Currently, there is no available non-invasive tool to assist in the identification of the aggressive form of this disease. Cell-free nucleic acid has been tested as a potential new molecular tool that can be useful in the diagnosis and prognosis of prostate cancer. MicroRNAs are a class of non-coding RNAs, that are shown to impact the development and progression of prostate cancer and their detection in plasma have been indicated as a promising non-invasive tool for disease screening and prognosis assessment. Among the several deregulated miRNAs that are observed in prostate cancer, the miRNA-141-3p was observed with upregulated expression levels in prostate cancer, particularly in the metastatic lesions. Therefore, the main aim of this study was to verify the diagnostic potential of miR-141 as a circulating tumor marker for prostate cancer and determine its functional role in modulating tumorigenesis in prostate cancer metastatic cell lines. MiR-141 expression analysis was performed in plasma samples of 102 prostate cancer patients without treatment and of 50 health controls by RT-qPCR. The experimental assays were performed by transfecting the PC3 cell line using miRNA-141 mimic/ inhibitor systems and directly accessing their effect in classical tumor phenotypes. Expression of the miR-141 were found significantly upregulated in the metastatic patients’ plasma specimens (FC=9.11, p=0.04; AUC=0.66) when compared to the patients without metastasis, showing its screening potential for prostate cancer metastasis. The in vitro ectopic expression of miR-141 in PC3 cell line showed a significant increased in cell proliferation, associated with changes in cell cycle (decreased number of cells in the G1 phase). Conversely, the inhibition of miR-141 showed an increase in the migratory ability, cell adhesion and in the Docetaxel cytotoxicity of the PC3 cells. Western-blot analysis showed that the inhibition of the miR-141 levels reduced the expression of the EMT inducers markers E-CADHERIN and CLAUDIN, and up-regulated the levels of ZEB-1, pAKT and VIMENTIN, consistent with their repression roles in the EMT process. These findings showed that miR-141 presents a direct function in controlling some of the in vitro metastatic phenotypes in prostate cancer, through the regulation of the epithelial-mesenchymal transition expression proteins. In conclusion, miR-141 presents a potential use as a minimally invasive molecular marker for prostate cancer diagnosis and can be used as a druggable target for therapy, particularly in the metastatic clinical setting. Citation Format: Marilesia Ferreira de Souza, Ilce Mara de Syllos Cólus, Aline Simoneti Fonseca, Hellen Kuasne, Paulo Emilio Fuganti, Deepak Kumar, Silvia Regina Rogatto, Luciane Regina Cavalli. Cell-free miR-141 as a molecular marker for prostate cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1476. doi:10.1158/1538-7445.AM2017-1476

Metabolomics studies of prostate cancer using gas chromatography-mass spectrometery

Background: Metabolomics is a powerful analytical approach that is widely used for early diagnosis of many tumors. In this study, we performed a comparative metabolomics analysis to identify metabolic differences in prostate cancer. Methods: We applied gas chromatography-mass spectrometry (GC-MS) to analyze the metabolic differences between normal prostate epithelial cells and prostate cancer cells (PC-3, LNCaP, and DU145 cells). We performed pairwise comparisons of the metabolomes for the intracellular and extracellular fluid using principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and Student’s t-test. Results: Our results revealed significantly different expression patterns among the metabolite groups, including amino acids, organic acids, sugars, and cholesterol. Of these four categories, cholesterol showed potential for combination with prostate-specific antigen (PSA) to improve diagnosis of prostate cancer. Conclusions: GC-MS may be an effective approach to reduce the frequency of invasive biopsies. Analysis of the diagnostic significance of prostate cancer markers will help to identify more meaningful tumor markers for prostate cancer diagnosis and treatment, contribute to the understanding of the pathogenesis of prostate cancer, and provide the basis for new ideas.

An original patient-derived xenograft of prostate cancer with cyst formation.

The high rate of failure of new agents in oncology clinical trials indicates a weak understanding of the complexity of human cancer. Recent understanding of the mechanisms underlying castration resistance in prostate cancer led to the development of new agents targeting the androgen receptor pathway; however, their effectiveness is limited. Hence, there is a need for experimental systems that are able to better reproduce the biological diversity of prostate cancer in preclinical settings. In this study, we established a unique patient-derived xenograft (PDX) model to identify biomarkers for treatment efficacy and resistance and better understand prostate cancer biology. A prostate cancer tissue sample from a Japanese patient was transplanted subcutaneously into male, severe combined immune-deficient (SCID) mice and this PDX mouse model was named KUCaP3. Sequential tumor volume changes were observed before and after castration. Androgen receptor (AR), prostate-specific antigen (PSA), and other molecular markers were examined immunohistochemically. Sequence analysis of AR was also performed to detect mutations. Proteomic analysis of cyst fluid and sera samples of KUCaP3 mice were analyzed by mass spectrometry (MS). KUCaP3 cell line, derived from human tissue, was successfully and serially passaged in vivo with approximately 60% take rate. KUCaP3 exhibited cyst formation, showed androgen-dependent growth initially, and developed castration-resistant growth several months after castration of the mice. Immunohistochemical analysis showed that KUCaP3 was positive for AR, PSA, CK18, and α-methyl acyl-coenzyme A racemase, but negative for CK5/6 and ERG. The AR gene in KUCaP3 cells contained a substitution from CAT (histidine) to TAT (tyrosine) at the nucleotide positions corresponding to codon 875 (H875Y) in the ligand-binding domain. Chemiluminescent immunoassay revealed higher levels of PSA in cystic fluid and the serum of KUCaP3-bearing mice. MS analysis detected 23 proteins of human origin in cystic fluids of KUCaP3. We developed KUCaP3, an androgen-dependent PDX model with cyst formation. Several proteins including PSA were detected in the cystic fluid and sera of tumor-bearing mice. This original PDX model has the potential to be used as a clinically relevant model to evaluate molecular markers for prostate cancer diagnosis and treatment. Prostate 76:994-1003, 2016. © 2016 Wiley Periodicals, Inc.

Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI.

Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd(3+) contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd(3+) binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 ± 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 ± 0.1 × 10(-22) M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM(-1) s(-1) and r2 of 37.9 mM(-1) s(-1) per Gd (55.2 and 75.8 mM(-1) s(-1) per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM(-1) s(-1) per Gd (188.0 mM(-1) s(-1) per molecule) and r1 of 18.6 mM(-1) s(-1) per Gd (37.2 mM(-1) s(-1) per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.

235P - Panel of Molecular Markers for Prostate Cancer Diagnosis

ABSTRACT Aim: The most important changes on molecular level affiliated by prostate cancer are epigenetic alterations of genetic materials including aberrant DNA methylation. We have investigated a panel of molecular markers of DNA methylation GSTp1, RARb2 and RASSF1A in order to create a test-system for diagnosis and management of prostate cancer. Methods: 157 patients in age 55-70 years (67,6 ± 7,7) with PSA level 4-10 ng/ml were included in our prospective study. Patients with prostatic cancer were also subdivided according to their Gleason score, PSA, age and TNM Staging. Blood, urine samples collected after digital rectal examination (DRE) and prostate tissue received by biopsy were used like biological materials. We assessed methylation of GSTp1, RARb2 and RASSF1A using a methylation-specific PCR assay and analyzed the sensitivity, specificity, positive predictive value and negative predictive value of each assay for the detection of genetic or epigenetic changes in circulating DNA. Results: Analyzed diagnostic characteristics of a molecular markers` panel calculated on batch of DNA samples excreted from prostate tissue, test-sensitivity was 86.3%, specificity was 76.6%. Sensisitivity of markers calculated on batch of DNA samples excreted from urine was 72.5% and specificity was 44.6%. Sensitivity of markers calculated on batch of DNA samples excreted from blood was 67.1% and specificity was 52.2%, from lymphocytes - 66% and 61.7% respectively. Statistical analysis did not reveal a significant correlation between panel of markers hypermethylation and Gleason score, PSA, age or TNM staging. Conclusions: Our data demonstrated the existence of significant differences in specificity between our diagnostic panel of markers and PSA. Specificity of markers GST p1, RARb2 and RASSF1A exceed specificity of PSA in «gray» zone (4-10 ng/ml) (61.7% vs 17%, р Disclosure: All authors have declared no conflicts of interest.

Reducing prostate cancer racial disparity: evidence for aggressive early prostate cancer PSA testing of African American men.

There is continuing controversy about prostate cancer testing and the recent American Urological Association guidelines. We hypothesize that the reduction and elimination of racial survival disparity among African American men (AAM; high-risk group) compared with European American men (EAM; intermediate-risk group) during the PSA testing era compared with the pre-PSA era strongly supports the use of PSA testing in AAM. We used Surveillance, Epidemiology, and End Results (SEER) data to investigate relative survival disparities between AAM and EAM. To evaluate pre-PSA testing era, we selected malignant first primary prostate cancer in AAM and EAM, all stages, diagnosed during 1973-1994. To evaluate relative survival disparities in the current PSA testing era, we selected malignant first primary local, regional, and distant stage prostate cancers diagnosed during 1998-2005 to calculate 5-year relative survival rates. Age-adjusted 5-year relative survival of prostate cancer diagnosed during 1973-1994 in the national SEER data revealed significantly shorter survival for AAM compared with EAM (P < 0.0001). The SEER-based survival analysis from 1995 to 2005 indicated no statistical difference in relative survival rates between AAM and EAM by year of diagnosis of local, regional, or distant stage prostate cancer. We conclude that the elimination of prostate cancer racial disparity of local, regional, and metastatic prostate cancer relative survival in the current PSA testing era compared with pre-PSA era as an endpoint to test PSA efficacy as a marker for prostate cancer diagnosis is evidence for aggressive testing of AAM. Evidence for screening AAM.

A DNA hypermethylation profile reveals new potential biomarkers for prostate cancer diagnosis and prognosis.

DNA hypermethylation has emerged as a novel molecular biomarker for the evaluation of prostate cancer diagnosis and prognosis. Defining the specific gene hypermethylation profile for prostate cancer could involve groups of genes that specifically discriminate patients with indolent and aggressive tumors. Genome-wide methylation analysis was performed on 83 tumor and 10 normal prostate samples using the GoldenGate Methylation Cancer Panel I (Illumina, Inc.). All clinical stages of disease were considered. We found 41 genes hypermethylated in more than 20% of the tumors analyzed (P < 0.01). Of these, we newly identified GSTM2 and PENK as being genes that are hypermethylated in prostate cancer and that were simultaneously methylated in 40.9% of the tumors analyzed. We also identified panels of genes that are more frequently methylated in tumor samples with clinico-pathological indicators of poor prognosis: a high Gleason score, elevated Ki-67, and advanced disease. Of these, we found simultaneous hypermethylation of CFTR and HTR1B to be common in patients with a high Gleason score and high Ki-67 levels; this might indicate the population at higher risk of therapeutic failure. The DNA hypermethylation profile was associated with cancer-specific mortality (log-rank test, P = 0.007) and biochemical recurrence-free survival (log-rank test, P = 0.0008). Our findings strongly indicate that epigenetic silencing of GSTM2 and PENK is a common event in prostate cancer that could be used as a molecular marker for prostate cancer diagnosis. In addition, simultaneous HTR1B and CFTR hypermethylation could help discriminate aggressive from indolent prostate tumors.

348 Panel of molecular markers for prostate cancer diagnosis

348 Panel of molecular markers for prostate cancer diagnosis

P060 Panel of molecular markers for prostate cancer diagnosis

P060 Panel of molecular markers for prostate cancer diagnosis

Cell-Free Circulating Plasma hTERT mRNA Is a Useful Marker for Prostate Cancer Diagnosis and Is Associated with Poor Prognosis Tumor Characteristics

BackgroundSerum prostate-specific antigen (PSA) is the most widely used marker for diagnosing prostate cancer (PCa). It lacks specificity and predictive value, resulting in inaccurate diagnoses and overtreatment of the disease. The aim of this study was to assess the usefulness of plasma telomerase reverse transcriptase (hTERT) mRNA as a diagnostic and prognostic tool for PCa and its association with clinicopathological parameters of tumors.Principal FindingsPlasma hTERT mRNA levels were determined by qRT-PCR in 105 consecutive patients with elevated PSA levels and in 68 healthy volunteers. The diagnostic accuracy, the efficacy as a prognostic factor of biochemical recurrence and the association with tumor clinicopathological parameters of plasma hTERT mRNA and serum PSA tests were determined using univariate and multivariate analyses. The results show that plasma hTERT mRNA is a non-invasive biomarker for PCa diagnosis that shows higher sensitivity (85% vs. 83%), specificity (90% vs. 47%), positive predictive value (83% vs. 56%), and negative predictive value (92% vs. 77%) than serum PSA. Plasma hTERT mRNA is significantly associated with poor prognosis tumor clinicopathological parameters and is a significant independent predictor of PCa (p<0.0001). Univariate analysis identified plasma hTERT mRNA (but not serum PSA) as a significant prognostic factor of biochemical recurrence. Plasma hTERT mRNA Kaplan-Meier curves confirmed the significant differences between groups and patients with higher levels than the cut-off value showed diminished recurrence-free survival (p = 0.004), whereas no differences were observed with serum PSA (p = 0.38). Multivariate analysis indicated that plasma hTERT mRNA (but not serum PSA) and stage were significantly associated with biochemical recurrence.ConclusionsOverall, these findings indicate that hTERT mRNA is a useful non-invasive tumor marker for the molecular diagnosis of PCa, affording a greater diagnostic and prognostic accuracy than the PSA assay and may be of relevance in the follow-up of the disease.

Immunoreactivity to caspase-3, caspase-7, caspase-8, and caspase-9 forms is frequently lost in human prostate tumors

Caspases are essential initiators and executioners of apoptosis. Changes in their expression may contribute to the development of proliferative disorders such as cancer, by altering the death-proliferation homeostatic balance. The aim of this work was to analyze the expression of a broad panel of caspases at the epithelial level in human prostate tissues to assess possible prostatic disease-related alterations. We comparatively analyzed by immunohistochemistry the expression of pro-caspase-3, pro-caspase-8, pro-caspase-9, cleaved caspase-3, cleaved caspase-8, and caspase-7, in normal and pathologic (benign hyperplasic, premalignant [high-grade intraepithelial neoplasia], and cancerous [prostate cancer]) human prostate epithelium. Expression of caspases was correlated with clinicopathologic features, including preoperative prostate-specific antigen levels, Gleason scores, and biochemical progression. Percentage of positive samples for all the analyzed caspases decreased in prostate cancer versus normal prostate epithelium. The values obtained for benign prostatic hyperplasia and high-grade intraepithelial neoplasia more qualitatively resembled those of the prostate cancer group. Our results indicate that caspase expression in prostate malignant cells is reduced in a substantial number of patients and that such an alteration occurs in the premalignant stage. Loss of caspase expression could constitute a useful marker for prostate cancer diagnosis. Therapeutic approaches aimed to recover or enhance caspase expression might be effective against prostate cancer.

Differential Protein Expression Profiling by iTRAQ-Two-dimensional LC-MS/MS of Human Bladder Cancer EJ138 Cells Transfected with the Metastasis Suppressor KiSS-1 Gene

KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumor stage, distant metastases, and poor overall survival in bladder tumors. To identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteomics analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full-length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24- and 48-h transfection were fractionated and cleaved with trypsin, and the resulting peptides were labeled with iTRAQ reagents. The labeled peptides were separated by strong cation exchange and reversed phase LC and analyzed by MALDI-TOF/TOF MS. Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology analysis, and Ingenuity Pathways Analysis to provide insight into biological networks. Comparative analysis among transfected, mock, and empty vector-exposed cells identified 1529 proteins with high confidence (>99%) showing high correlation rates among replicates (70%). The involvement of the identified proteins in biological networks served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for verification analyses by Western blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumors spotted onto tissue microarrays (n = 280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.

A Robust Error Model for iTRAQ Quantification Reveals Divergent Signaling between Oncogenic FLT3 Mutants in Acute Myeloid Leukemia

The FLT3 receptor tyrosine kinase plays an important role in normal hematopoietic development and leukemogenesis. Point mutations within the activation loop and in-frame tandem duplications of the juxtamembrane domain represent the most frequent molecular abnormalities observed in acute myeloid leukemia. Interestingly these gain-of-function mutations correlate with different clinical outcomes, suggesting that signals from constitutive FLT3 mutants activate different downstream targets. In principle, mass spectrometry offers a powerful means to quantify protein phosphorylation and identify signaling events associated with constitutively active kinases or other oncogenic events. However, regulation of individual phosphorylation sites presents a challenging case for proteomics studies whereby quantification is based on individual peptides rather than an average across different peptides derived from the same protein. Here we describe a robust experimental framework and associated error model for iTRAQ-based quantification on an Orbitrap mass spectrometer that relates variance of peptide ratios to mass spectral peak height and provides for assignment of p value, q value, and confidence interval to every peptide identification, all based on routine measurements, obviating the need for detailed characterization of individual ion peaks. Moreover, we demonstrate that our model is stable over time and can be applied in a manner directly analogous to ubiquitously used external mass calibration routines. Application of our error model to quantitative proteomics data for FLT3 signaling provides evidence that phosphorylation of tyrosine phosphatase SHP1 abrogates the transformative potential, but not overall kinase activity, of FLT3-D835Y in acute myeloid leukemia.

The value of combined detection of PCA3 mRNA and PSA mRNA in peripheral blood of patients with prostate cancer

Objective To evaluate the application value of combined detection of PCA3 and PSA mRNA in peripheral blood of patients with prostate cancer(PCa) for evaluation of mien)metastasis. Methods PCA3 and PSA mRNA were detected by duplex real time quantitative RT-PCR in a total of 49 PCa and 71 benign protatic hyperplasia (BPH) patients' peripheral blood. The diagnostic value was analyzed by receiver operative characteristic(ROC) curve. Results The levels of PCA3 mRNA in PCa patients were significantly higher than those in BPH patients [2 362( <30-7 421 ) copies/ml vs <30 copies/M, Z = -6. 66, P < 0. 01 ], and the same to PSA mRNA [3 425 ( 908-36 639 ) copies/ml vs < 200 copies/ml, Z = - 6. 40, P<0. 01 ]. The positive rate of PCA3 and PSA mRNA in peripheral blood was positively correlated with clinical stage[clinical stage B: 30.0% (3/10), C: 60.0% (9/15) and 86.7% (13/15), D: 91.7% (22/24) and 91.7% (22/24) ,Chi-square = 13. 534 and 16. 541, P <0. 01, respectively]. Meanwhile, the positive rate of PCA3 mRNA and PSA mRNA was also increased with the increase of Gleason score[ Gleason score of 2 to 4 : 20.0% (1/5) and 40. 0% (2/5) ;5 to 7 : 66.7% (12/18) and 72. 2% ( 13/18 ) ;8 to 10 : 84. 6% (22/26) and 92.3% ( 24/26 ) ;Chi-square = 8. 895 and 8. 015, P < 0. 05, respectively ]. ROC analysis showed that the sensitivities for PCA3 and PSA mRNA were 69. 4% (34/49) and 81.7% (40/49) and the specificities was 90. 1% (64/71) and 77.5% (55/71), respectively, when the cut-off value was 846 copies/ml for PCA3 mRNA and 280 copies/ml for PSA mRNA. Meanwhile, the sensitivity can reach to 85.7% (42/49) when the detection of PCA3 and PSA mRNA were combined. However, the specificity was decreased to 76. 1% (54/71). For the diagnosis of PCa micrometastasis, the sensitivity and specificity for PCA3 mRNA was 90.9% (20/22) and 84.7% (11/13), respectively. Conclusions PCA3 and PSA mRNA in peripheral blood are useful markers for PCa diagnosis. Simultaneous detection for PCA3 and PSA mRNA is more helpful for PCa diagnosis. Meanwhile, detection of PCA3 mRNA is a useful marker for diagnosing PCa micrometastasis. Key words: Prostatic neoplasms; Neoplasms metastasis; Antigens, neoplasm; Prostate-specific antigen; Reverse transcriptase polymerase chain reaction