Showing papers by "Carlos Cordon-Cardo published in 2006"

Defining Molecular Profiles of Poor Outcome in Patients With Invasive Bladder Cancer Using Oligonucleotide Microarrays

Abstract: Purpose Bladder cancer is a common malignancy characterized by a poor clinical outcome when tumors progress into invasive disease. We sought to define genetic signatures characteristic of aggressive clinical behavior in advanced bladder tumors. Methods Oligonucleotide arrays were utilized to analyze the transcript profiles of 105 bladder tumors: 33 superficial, 72 invasive lesions, and 52 normal urothelium. Hierarchical clustering and supervised algorithms were used to classify and stratify bladder tumors on the basis of stage, node metastases, and overall survival. Immunohistochemical analyses on bladder cancer tissue arrays (n = 294 cases) served to validate associations between marker expression, staging and outcome. Results Hierarchical clustering classified normal urothelium, superficial, and invasive tumors with 82.2% accuracy, and stratified bladder tumors on the basis of clinical outcome. Predictive algorithms rendered an 89%-correct rate for tumor staging using genes differentially expressed betw...

Identification of a tumour suppressor network opposing nuclear Akt function

Abstract: The proto-oncogene AKT (also known as PKB) is activated in many human cancers, mostly owing to loss of the PTEN tumour suppressor1. In such tumours, AKT becomes enriched at cell membranes where it is activated by phosphorylation. Yet many targets inhibited by phosphorylated AKT (for example, the FOXO transcription factors) are nuclear; it has remained unclear how relevant nuclear phosphorylated AKT (pAKT) function is for tumorigenesis. Here we show that the PMLtumour suppressor prevents cancer by inactivating pAKT inside the nucleus. We find in a mouse model that Pml loss markedly accelerates tumour onset, incidence and progression in Pten-heterozygous mutants, and leads to female sterility with features that recapitulate the phenotype of Foxo3a knockout mice2. We show that Pml deficiency on its own leads to tumorigenesis in the prostate, a tissue that is exquisitely sensitive to pAkt levels, and demonstrate that Pml specifically recruits the Akt phosphatase PP2a as well as pAkt into Pml nuclear bodies. Notably, we find that Pml-null cells are impaired in PP2a phosphatase activity towards Akt, and thus accumulate nuclear pAkt. As a consequence, the progressive reduction in Pml dose leads to inactivation of Foxo3a-mediated transcription of proapoptotic Bim and the cell cycle inhibitor p27kip1. Our results demonstrate that Pml orchestrates a nuclear tumour suppressor network for inactivation of nuclear pAkt, and thus highlight the importance of AKT compartmentalization in human cancer pathogenesis and treatment.

PML inhibits HIF-1α translation and neoangiogenesis through repression of mTOR

Abstract: Loss of the promyelocytic leukaemia (PML) tumour suppressor has been observed in several human cancers. The tumour-suppressive function of PML has been attributed to its ability to induce growth arrest, cellular senescence and apoptosis. Here we identify PML as a critical inhibitor of neoangiogenesis (the formation of new blood vessels) in vivo, in both ischaemic and neoplastic conditions, through the control of protein translation. We demonstrate that in hypoxic conditions PML acts as a negative regulator of the synthesis rate of hypoxia-inducible factor 1α (HIF-1α) by repressing mammalian target of rapamycin (mTOR). PML physically interacts with mTOR and negatively regulates its association with the small GTPase Rheb by favouring mTOR nuclear accumulation. Notably, Pml-/- cells and tumours display higher sensitivity both in vitro and in vivo to growth inhibition by rapamycin, and lack of PML inversely correlates with phosphorylation of ribosomal protein S6 and tumour angiogenesis in mouse and human tumours. Thus, our findings identify PML as a novel suppressor of mTOR and neoangiogenesis. The tumour suppressor PML ('promyelocytic leukaemia') is shown to be an inhibitor of blood vessel formation in tumours and in tissue recovering from ischaemia. Loss of PML is associated with a range of human cancers. Its role in angiogenesis control marks it as a drug target in cancers, where upregulation is the aim, and in encouraging blood vessel formation following ischaemic heart attack or stroke, by downregulation. The tumour suppressor gene PML is shown to normally restrain angiogenesis, both in tumours and under ischaemic conditions. This involves a new pathway in which during hypoxia, PML regulates mTOR and the transcription factor HIF-1α

Autocrine PDGFR signaling promotes mammary cancer metastasis

Abstract: Metastasis is the major cause of cancer morbidity, but strategies for direct interference with invasion processes are lacking. Dedifferentiated, late-stage tumor cells secrete multiple factors that represent attractive targets for therapeutic intervention. Here we show that metastatic potential of oncogenic mammary epithelial cells requires an autocrine PDGF/PDGFR loop, which is established as a consequence of TGF-β–induced epithelial-mesenchymal transition (EMT), a faithful in vitro correlate of metastasis. The cooperation of autocrine PDGFR signaling with oncogenic Ras hyperactivates PI3K and is required for survival during EMT. Autocrine PDGFR signaling also contributes to maintenance of EMT, possibly through activation of STAT1 and other distinct pathways. Inhibition of PDGFR signaling interfered with EMT and caused apoptosis in murine and human mammary carcinoma cell lines. Consequently, overexpression of a dominant-negative PDGFR or application of the established cancer drug STI571 interfered with experimental metastasis in mice. Similarly, in mouse mammary tumor virus–Neu (MMTV-Neu) transgenic mice, TGF-β enhanced metastasis of mammary tumors, induced EMT, and elevated PDGFR signaling. Finally, expression of PDGFRα and -β correlated with invasive behavior in human mammary carcinomas. Thus, autocrine PDGFR signaling plays an essential role during cancer progression, suggesting a novel application of STI571 to therapeutically interfere with metastasis.

Profiling bladder cancer using targeted antibody arrays.

Abstract: Bladder cancer is a common malignancy requiring a high degree of surveillance because of the frequent recurrences and the poor clinical outcome of invasive disease. To date, serum biomarkers for bladder cancer lack optimal sensitivity and specificity to assist in diagnosis and disease categorization. Here, we designed antibody arrays for bladder cancer by selecting antibodies against targets differentially expressed in bladder tumors. Serum protein profiles measured by an antibody array containing 254 antibodies discriminated bladder cancer patients from controls (n = 95) with a correct classification rate of 93.7%. A second independent antibody array containing 144 antibodies revealed that protein profiles provide predictive information by stratifying patients with bladder tumors (n = 37) based on their overall survival (P = 0.0479). In addition, serum proteins, such as c-met, that were top ranked at identifying bladder cancer patients were associated with pathological stage, tumor grade, and survival when validated by immunohistochemistry of tissue microarrays containing bladder tumors (n = 173). This study provides experimental evidence for the use of several integrated technologies strengthening the process of biomarker discovery. Serum protein profiles obtained by antibody arrays represent comprehensive means for bladder cancer diagnosis and clinical outcome stratification, which could potentially assist in selection of cancer patients who would benefit from early, individualized therapeutic intervention.

Amplification of CDK4 and MDM2 in malignant melanoma.

Abstract: Amplification of the 12q13-15 region is a common event in several human tumors including liposarcomas, gliomas, and osteosarcomas. We have demonstrated high-level amplification of 12q14 in a subset of uncultured malignant melanomas (3 of 53). High-resolution mapping of the amplicon using quantitative PCR revealed a bipartite amplicon consisting of a primary 50-kb amplicon centered on CDK4 and a secondary amplicon centered on MDM2, without amplification of the intervening 11 Mb of genomic DNA. Analysis of mRNA and protein levels in melanomas with 12q14 amplification demonstrated overexpression of target genes CDK4 and MDM2 without loss of CDKN2A-P16 (P16INK4A) or CDKN2A-P14ARF (P14ARF) expression, important regulators of the RB1 and TP53 pathways, which are commonly lost or mutated in melanoma. These results suggest that coamplification of CDK4 and MDM2 may substitute for loss of P16INK4A and P14ARF function in a subset of melanomas.

Determinants of sensitivity and resistance to rapamycin-chemotherapy drug combinations in vivo.

Abstract: The phosphatidylinositol-3-OH kinase [PI(3)K] pathway is frequently activated in human cancers and represents a rational target for therapeutic intervention. We have previously shown that enforced expression of Akt, which is a downstream effector of PI(3)K, could promote tumorigenesis and drug resistance in the Emu-myc mouse lymphoma model, and that these tumors were particularly sensitive to inhibition of mammalian target of rapamycin (mTOR) with rapamycin when combined with conventional chemotherapy. We now show that reduced dosage of PTEN, a negative regulator of PI(3)K signaling, is sufficient to activate Akt, but has only a modest effect on lymphomagenesis in the same model. Nonetheless, loss of even one PTEN allele resulted in lymphomas that were resistant to conventional chemotherapy yet sensitive to rapamycin/chemotherapy combinations. These effects could be recapitulated by using RNA interference to suppress PTEN expression in lymphomas, which were previously established in the absence of PI(3)K lesions. Finally, the introduction of lesions that act downstream of mTOR (eIF4E) or disable apoptosis (Bcl-2 and loss of p53) into PTEN+/- lymphomas promoted resistance to rapamycin/chemotherapy combinations. Thus, whether activation of the PI(3)K pathway confers sensitivity or resistance to therapy depends on the therapy used as well as secondary genetic events. Understanding these genotype-response relationships in human tumors will be important for the effective use of rapamycin or other compounds targeting the PI(3)K pathway in the clinic.

Apaf-1 expression in malignant melanoma.

Abstract: We have previously reported that 10 out of 19 metastatic melanoma cell lines and 10 out of 24 melanoma specimens expressed low levels of Apaf-1, and that Apaf-1 expression inversely correlated with the chemosensitivity of melanoma cell lines in vitro. Based on these observations, we proposed that Apaf-1 downregulation contributes to chemoresistance in malignant melanoma. In that study, we also suggested that Apaf-1 downregulation may be a late event during tumor progression (four out of five in situ melanomas were positive for Apaf-1). However, two recent Letters to the Editor published in this journal by Allen et al. and Peltenburg et al. imply a lower rate of downregulation of Apaf-1 in melanoma. In their panel of metastatic melanoma cell lines, only 1/11 and 1/10, respectively, were negative for Apaf-1. What could explain the dissimilar rates of Apaf-1 repression between studies? One possibility is methodological. However, the antibody used in our study is specific – it recognizes a polypeptide that has the electrophoretic mobility of Apaf-1, associates with caspase-9 in cell free systems, and is lost from cells transfected with two different siRNAs against Apaf-1. Moreover, our experiments correlating Apaf-1 expression with chemosensitivity were conducted blindly – one laboratory (YL) measured Apaf-1 expression and the other (SWL) assayed apoptosis and the results were compared later. Finally, we confirmed the immunoblotting results by Northern blotting and in situ hybridization and, more recently, other investigators corroborated our observations using three different antibodies, and by LOH analyses and quantitative RT-PCR. Given that technical issues are unlikely to account for the differences between data sets, it is possible that the studies by Peltenburg and Allen did not examine enough samples to make reliable conclusions. In fact, six recent studies that analyzed over 400 pigmented lesions support the downregulation of Apaf-1 during melanoma progression. Fujimoto et al. concluded that one copy of the locus containing the Apaf-1 gene is lost in 37% of metastatic melanomas, and this deletion can also be detected in circulating cells. mRNA expression studies supported the impact of deletions in this locus on Apaf-1 expression. Baldi et al., Dai et al., Zannon et al. and Mustika et al. report a heterogeneous expression of Apaf-1, highly positive in benign nevi but weak or undetectable in 35–50% in situ melanomas and lymph node and visceral metastases. In addition, Qin et al. have also independently reported low Apaf-1 expression in metastatic melanoma cell lines used in our initial study. These results, along with recent reports of low Apaf-1 expression in other melanoma cell lines, are consistent with our initial findings. Still, it is conceivable that there are bona fide differences in Apaf-1 expression between melanoma cell lines depending on their site of origin or culture conditions. This would not be surprising, as melanoma is notorious for its heterogeneity and, accordingly, such discrepancies have been reported before. For example, BRAF was originally found mutated in 66% of malignant melanomas, but subsequently rates varying from 0 to 100% have been observed depending on the anatomical site and association with sun exposure. Also, most studies find that p53 is rarely mutated in melanoma; however, Peltenburg and co-workers have reported that p53 mutations can occur in up to 44% of melanomas derived from chronically sun-exposed sites. Alternatively, the mechanisms repressing Apaf-1 in primary tumor specimens can be occasionally altered through genetic or epigenetic changes that accompany passaging in culture (e.g., by potentiation of the Rb/E2F-1 pathway, which can control Apaf-1 mRNA levels). The fact that certain leukemia cells as well as AML, ALL, and CML blasts and cells from glioblastoma and cervical cancer may also inactivate Apaf-1 expression, in part by methylation, suggests a broader impact of tumorrelated events modulating Apaf-1 expression. We agree with Allen and Peltenburg that Apaf-1 downregulation is not the only determinant of chemoresistance in melanoma, and highlighted a drug-resistant line (SK-Mel-173) that retained high Apaf-1 expression in our initial report. Nonetheless, several studies suggest that Apaf-1 levels can affect apoptosis in melanoma cells. We showed that reexpression of Apaf-1 in cells with low Apaf-1 levels restored the ability of doxorubicin (adriamycin) to efficiently induce apoptosis, and this observation was independently confirmed and extended to cisplatin and vinblastin in isogenic settings. Similarly, Furukawa et al. showed that melanoma cells expressing low levels of Apaf-1 are resistant to apoptosis induced by E2F-1. In contrast, Peltenburg et al. did not find a direct correlation between Apaf-1 levels and caspase-9 activity in response to etoposide. Intriguingly, these results of Peltenburg et al. are in contrast to a recent report showing a delayed or increased resistance of low Apaf-1-expressing melanoma cells to high doses of etoposide (as well as doxorubicin or 5FU). In this context, it should be noted that the Peltenburg group has already reported mechanistic variability in the response of melanoma cells to etoposide. In summary, while it is clear that Apaf-1 loss does not universally promote cell survival and is not the sole determinant of melanoma chemoresistance, we think it premature to minimize its role in melanoma without further functional studies. Like other apoptotic regulators, its role in Cell Death and Differentiation (2006) 13, 352–353 & 2006 Nature Publishing Group All rights reserved 1350-9047/06 $30.00

p53 restoration in liver carcinomas induces cellular senescence and tumor clearance through an innate immune response

Abstract: 4913 Although cancer arises from a combination of mutations in oncogenes and tumor suppressor genes, the extent to which tumor suppressor gene loss is required for the maintenance of established tumors is poorly understood p53 is an important tumor suppressor, whose mutation produces defects in cell cycle checkpoints, senescence, and apoptosis, and promote angiogenesis and genomic instability1-3 To determine the consequences of p53 reactivation in vivo, we used RNA interference to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma We show that even brief reactivation of endogenous p53 in p53-deficient tumors can produce complete tumor regressions The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the up-regulation of inflammatory cytokines This program, while producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumor cells in vivo, thereby contributing to tumor clearance Our study indicates that p53 loss is required for the maintenance of aggressive carcinomas, and establishes how a cellular senescence program can act together with the innate immune system to potently limit tumor growth

Joint association of polymorphism of the FGFR4 gene and mutation TP53 gene with bladder cancer prognosis.

Abstract: The impact of the fibroblast growth factor receptor 4 (FGFR4) Gly388Arg polymorphism on bladder cancer is unknown. We found no clear correlations between the FGFR4 genotype and risk of bladder cancer or pathological parameters. Neither the polymorphism nor TP53 mutation status was an independent predictor of prognosis, but they might act jointly on the disease-specific survival of patients.

Altered expression of DNA double-strand repair genes Ku70 and Ku80 in carcinomas of the oral cavity.

Abstract: The relevance of double-strand DNA repair genes has been demonstrated in several tumours. The main aim of this study was to analyse the expression of the heterodimers Ku70 and Ku80, building regulatory subunits of the DNA-dependent protein kinase in 40 oral carcinomas. Ku70 expression was found in 87.5% of grade 1 and grade 3 tumours and in 82.9% of grade 2 carcinomas. Ku80 presence was noted in 87.5% of grade 1 tumours, 82.9% of grade 2 tumours and in all grade 3 tumours. Ku70-positive cells were present in 90.5% of tumours without and in 80% of tumours with lymphatic metastases. A similar relationship was found for Ku80 expression. Additionally, the expression of Ku70 was highly significantly related to smoking habits. Our results demonstrated that defects of DNA double-strand repair genes play an important role in the tumour progression of oral carcinomas.

Evaluation of Ki67, p53 and angiogenesis in patients enrolled in a randomized study of neoadjuvant chemotherapy with or without cystectomy: a Southwest Oncology Group Study.

Abstract: In this prospective biomarker study, we evaluated the prognostic significance of Ki67, p53 and angiogenesis in patients with locally advanced bladder cancer. The patients were volunteers from a Southwest Oncology Group trial of locally advanced bladder cancer who were randomized to treatment with neoadjuvant chemotherapy plus cystectomy or cystectomy alone. Tissue specimens were obtained prior to neoadjuvant chemotherapy from 42 patients randomized to receive the combination-treatment arm and 52 randomized to cystectomy alone. The statistical power of the study was quite limited by the small sample size. The biomarkers were assayed by immunohistochemistry. Angiogenesis was determined using anti-CD34 immunostaining. Patients whose tumors had increased Ki67 expression had better progression-free survival that was marginally significant, p=0.063. The median survival in those with higher Ki67 expression was 73 months, and in those with lower expression was 38 months. However, this did not achieve statistical significance, p=0.25. There was a suggestion of worse survival among patients whose tumors exhibited altered p53 staining [hazard ratio (HR) = 1.48; p=0.15], but there was no difference in progression-free survival (HR=1.02; p=0.93). The enumeration of tumor microvessels did not provide prognostic information.

Analysis of DNA Mismatch Repair Gene Expression and Mutations in Thyroid Tumours

Abstract: Alterations of DNA mismatch repair genes, primarily demonstrated in hereditary nonpolyposis colorectal carcinomas, were reported to be of relevance for the progression of several sporadic tumours. In this study, the expression and mutations of MLH1, MSH2, PMS1 and PMS2 in a panel of thyroid tumours, including nodular hyperplasia, follicular adenomas and carcinomas, were investigated. The expressions of MLH1, MSH2 and PMS1 were generally higher in malignant tumours than in benign lesions (p<0.01). This observation can find potential diagnostic application in the differentiation of follicular adenomas from follicullar carcinomas of the thyroid. No point mutations in the DNA mismatch repair genes MSH2 (exon 12, 13) and MLH1 (exon 15, 16) were found. DNA mismatch repair has been shown to play a very important role in the prevention of genetic instability. The defects of this system were first described in HNPCC (hereditary nonpolyposis colorectal carcinoma) (1-7) and later were also demonstrated in several sporadic tumours including melanomas, bladder cancer and breast tumours (8-12). For humans, four genes, namely MLH1, MSH2, PMS1 and PMS2, are responsible for the control of DNA mismatch repair. It is possible to immunohistochemically investigate the expression of these genes at the protein level. Thyroid tumours have not been investigated in terms of the state of the DNA mismatch repair proteins. Microsatellite instability and some LOH have been found in thyroid carcinomas (13, 14). Accepting microsatellite instability as the result of malfunctioning DNA mismatch repair, a working hypothesis that some alterations of DNA mismatch repair protein expression and eventual mutations in DNA mismatch repair genes are found in thyroid carcinomas can be formulated. Deletion of the short arm of chromosome 3, including the locus of MLH1, reported for thyroid carcinomas, was an additional argument in favour of this hypothesis (15). The overall target of this study was to check our working hypothesis and additionally to judge the diagnostic relevance of DNA mismatch repair gene expression.

Altered patterns of RB expression define groups of soft tissue sarcoma patients with distinct biological and clinical behavior.

Abstract: Summary. Background: Function of the retinoblastoma tumor suppressor protein (pRB) may be compromised at a genetic level by gene loss or mutation or at a posttranslational level by hyperphosphorylation. In this study, we examined adult soft tissue sarcomas (ASTS) to determine if alterations of pRB were associated with distinct patterns of pRB expression and clinical outcome. Design: We investigated 86 ASTS patients using monoclonal antibodies that distinguish between hyperphosphorylated and underphosphorylated pRB products. We also used microsatellite analysis to investigate the genetic status of the RB locus. We correlated pRB alterations with proliferative activity, and with clinicopathological outcomes. Results: Altered patterns of pRB expression are common in ASTS occurring in 84% of cases, and it is significantly associated with proliferative activity (p<0.001). Patients whose tumors either lack expression of pRB, or express hyperphosphorylated forms of pRB, have poor survivals compared to patients whose tumors exhibit a normal, underphosphorylated pattern of pRB expression (p=0.03). In addition, 63% of cases lacking expression of pRB showed loss-of-heterozygosity at the locus. Conclusions: Inactivation of pRB is common in adult STS, which may be due to either gene loss or posttranslational modification, namely hyperphosphorylation. Both mechanisms are associated with tumor cell proliferation and poor survival.

Exonic Deletions of Mismatch Repair Genes MLH1 and MSH2 Correlate with Prognosis and Protein Expression Levels in Malignant Melanomas

Abstract: The mutations of MLH1 and MSH2 have been reported to be responsible for malignant transformation and tumour progression in several sporadic tumours. Eighty-six primary malignant melanomas with known follow-up were investigated. Point mutations of DNA mismatch repair MLH1 and MSH2 in malignant melanomas were not found. Exon 12 (MSH2) was not present in 26 out of the 86 melanomas and exon 13 (MSH2) was lost in 25 of the tumours. The loss of exon 15 (MLH1) was observed in 22 out of the 86 tumours and the loss of exon 16 (MLH1) in 24 melanomas. The loss of exons correlated strongly with the loss of MLH1 and MSH2 protein expression. In multivariate analysis, including all 4 exons and expressions of MLH1 and MSH2, prognostic significance was found only for loss of exon 12 (MSH2) and loss of exon 15 (MLH1).

Alterations of the retinoblastoma and p16 pathway correlate with promoter methylation in malignant fibrous histiocytomas.

Abstract: Recent reports indicate that the alterations in the p16 and pRb pathways can influence tumour progression and poor prognosis in several tumours. The objective of this study was to analyse p16 and pRb expression in161 patients with malignant fibrous histiocytomas (MFH). By immunohistochemistry, p16 and pRb were demonstrated in 25% and 56% of MFH, respectively. Cox regression analysis demonstrated an independent prognostic influence of both genes. Generally, the loss of p16 and pRb expression correlated with poorer prognosis. Promoter methylation of p16 was found in 16/42 of p16 negative MFH and of pRb in 2/42 of pRb-negative MFH. It can be concluded that p16 and pRb alterations play an important role in the progression of soft tissue sarcomas.

A putative tumor suppressor role for Wnt-signaling in sarcomagenesis

Abstract: 9507 Background: We sought to elucidate the relationship between the human adult mesenchymal stem cell (hMSC), Wnt signaling and sarcomagenesis. Methods: In vitro hMSC differentiation, microarray g...

Tumor-specific serum peptidome patterns.

Abstract: Proc Amer Assoc Cancer Res, Volume 47, 2006 SY05-01 Most anti-cancer strategies rely on early detection, followed by close monitoring for early relapse so that therapies can be appropriately adjusted There is optimism, however, that recent advances in genomics and proteomics may more readily lead to new and improved approaches in molecular diagnostics, capable of classifying patients into subgroups based on predicted response to individual treatment Biomarker based screens should be minimally invasive and reproducible For instance, a simple blood or urine test that detects molecules specific to tumor tissues would be ideal In addition, screening technology should be sufficiently sensitive to detect early cancers but specific enough to classify individuals without cancer as being free of disease While genes contain the hereditary information, including genetic predisposition to cancer and other diseases, it is their products that confer the actual phenotypes of living organisms and, in case of disease, normal versus pathological states Since there are many post-translational events that can modify the structure, function, and degradation of proteins, the knowledge of genes alone does not even begin to describe the full complexity of biological systems From a screening perspective, it is also mainly the proteins that are secreted or otherwise released from tissues into the blood stream Yet, despite an intensive search during the past decade(s), only a small number of identified cancer biomarkers, all plasma proteins, have proven clinically useful, often in combination with other diagnostic tools, for the prognosis of response to therapy, relapse and survival, for defining the rate of progression and monitoring of treatment, but less so for broad based population screening Those proteins are typically present in plasma or serum at sub-nanomolar concentrations and require individual immuno-assays for detection and quantitation New and improved cancer biomarkers and facile detection methods are clearly on order but have so far eluded discovery and implementation Even the most recent approaches, using identity-based proteomics that involves digesting (eg, with trypsin) complex protein mixtures into peptides for mass spectrometric (MS) analysis, have yet to translate into any practical applications As cancer involves the transformation and proliferation of altered cell types that produce high levels of specific proteins and enzymes such as proteases (eg, PSA and PSMA), it will not only modify the array of existing serum proteins (serum proteome) but also their metabolic products, ie peptides (serum peptidome) It is well established that human serum contains thousands of proteolytically derived peptides, yet it remains unclear to date whether this complex peptidome may provide a correlate of some biological events occurring in the entire organism As advances in MS permit the display of hundreds of small to medium sized peptides using microliter volumes of serum, several researchers have advocated the use of MS-based serum peptide profiling to determine qualitative and quantitative patterns, often referred to as signatures or barcodes, that indicate the presence / absence of disease However, this work has come under criticism as growing evidence indicated that uncontrolled variables related to both clinical and analytical chemistry, and/or signal processing artifacts, may have tainted the published results In any case, the identities of only a few putative markers have been established so far Yet, the proof of the potential value of this new approach will be in the ability of several laboratories to independently show that the discriminatory peptides have the same amino acid sequences Working towards this goal, we have developed an automated procedure for the simultaneous measurement of peptides in serum that utilizes magnetic, reversed-phase beads for analyte capture and a MALDI-TOF MS read-out This system is more sensitive than surface capture on chips as spherical particles have larger combined surface areas, and therefore higher binding capacity, than small-diameter spots Coupled to high-resolution MS and MS/MS, hundreds of peptides can be detected in a droplet of serum, many of which can be readily identified without further fractionation The automation facilitates throughput and ensures reproducibility Furthermore, we have developed a minimal entropy-based algorithm that simplifies and improves alignment of spectra and subsequent statistical analysis With these tools in hand, we have sought to determine if selected patterns of serum peptides with known sequences can (i) separate cancer from non-cancer, (ii) distinguish between different types of solid tumors, and (iii) allow class prediction with an independent validation set We used peptide-ion relative intensity comparisons, statistical analysis and visual inspection of spectral overlays to sort through hundreds of features obtained by rigorous peptide profiling of 106 serum samples from patients with advanced prostate, or bladder or breast cancer, and from healthy controls, to identify several that are most predictive of outcome It appeared that reduction in the number of key peptides to only a few (ie the signatures or barcodes) that are easily recognized between samples did not adversely affect class predictions Targeted MS/MS-based sequence-identification of 61 barcode-peptides indicated that all were breakdown products, many related, of abundant proteins in the blood By correlating the proteolytic patterns with disease groups and controls, we show that exoprotease activities, superimposed on the ex-vivo coagulation and complement-degradation pathways, contribute to generation of not only cancer-specific but also cancer type-specific serum peptides This small but robust set of marker peptides then enabled highly accurate class prediction for an external validation set of prostate cancer samples Our study therefore provides a direct link between peptide marker profiles of disease and differential protease activity The patterns we describe may have clinical utility as surrogate markers for detection and classification of cancer Our findings have also important implications for future peptide biomarker discovery efforts

Systems pathology for building predictive models: The androgen receptor as a prototype biomarker in prostate cancer progression and targeted therapeutic response assessment

Abstract: 4504 Background: A functional androgen receptor (AR) signaling axis plays a critical role in prostate cancer (PCA) development and progression across the clinical spectrum of the illness. Following...