Cytosine methyltransferases as tumor markers

Changes in DNA methylation patterns is a prominent characteristic of human tumors. Tumor cells display reduced levels of genomic DNA methylation and site-specific CpG island hypermethylation. Methylation of CpG dinucleotides is catalyzed by the enzyme family of DNA methyltransferases (DNMTs). In this review, the role of DNA methylation and DNMTs as key determinants of carcinogenesis is further elucidated. The chromatin modifying proteins that are known to interact with DNMTs are also described. Finally, the role of DNMTs as potential therapeutic targets is addressed.     

Urinary modified nucleosides as tumor markers

Extracts of urinary nucleosides have been sequentially purified and examined by mass spectrometric analysis. Seventeen modified nucleosides have been unequivocally identified and a further five provisionally identified. While several nucleosides were found only in a small number of extracts, the occurrence and levels of others were found to correlate with the tumour type and stage.     

Genetic defects as markers of tumor development

Carcinogenesis is long-term multistep accumulation of defects of genes responsible for cell division, DNA repair, and apoptosis. The functions of these genes are known both for norm and for pathologies caused by their damage and resulting in "asocial" cell behavior. Owing to the recent progress in studying the mechanisms of carcinogenesis, some genetic defects may be considered from the applied point of view (as tumor markers rather than as pathogenetic factors) and employed in diagnostics. Thus detection of mutant alleles in biological fluids (e.g., beyond the tumor) suggests higher risk of carcinogenesis. Genetic defects are a new class of tumor markers and have a substantial diagnostic potential. In contrast to known protein markers (alpha-fetoprotein, etc.) used in clinical practice, DNA markers are oncospecific (as these are in direct cause-and-effect relationships with carcinogenesis) and universal (as there is not a single tumor cell without a genetic defect). Analysis of DNA markers may be employed not only in diagnostics or tumor growth monitoring (assessment of treatment efficiency, early detection of recurrence or metastasis), but also (prospectively) in screening (tumor detection at the presymptomatic stage, identification of high-risk groups). Theoretical grounds, prospects, problems, and methods of this new field are considered.     

Fluorophor-labeled spermidine derivatives as fluorescent markers in optical tumor imaging

Up-regulation of polyamine transporters on the surface of tumor cells and the internalization of biogenic polyamines by active transport processes may be exploited for the accumulation of spermidine derivatives as reporter molecules. We have synthesized and tested fluorophor-labeled spermidine derivatives for the development of a new class of intraoperative tumor imaging agents. In vitro uptake experiments and initial in vivo imaging studies illustrated that fluorophor tagged spermidine derivatives show tumor accumulation.     

Diacetylated derivatives of spermine and spermidine as novel promising tumor markers

N1,N12-diacetylspermine (DiAcSpm) and N1,N8-diacetylspermidine (DiAcSpd) are minor components of human urinary polyamine to which little attention has been paid until recently. HPLC analysis of urinary polyamines has revealed that the excretion of these diacetylpolyamines, in particular, into urine was frequently and markedly increased in association with every type of cancer so far examined. Remission was usually accompanied by recovery of urinary diacetylpolyamines to the normal level. DiAcSpm was more sensitive than CEA for detecting colorectal cancer patients, while DiAcSpd was highly specific for malignant conditions in that the excretion of the latter was scarcely elevated in cases of benign urogenital diseases. An ELISA procedure for rapid determination of DiAcSpm was developed to promote the clinical application of these new tumor markers, and subsequent studies indicated that DiAcSpm was elevated in 60% of colorectal cancer patients at early stages (stage 0 + I), whereas only 10% of these patients were CEA-positive. DiAcSpm may also be useful as a follow-up marker that is efficient for detecting recurrence and sensitive to changes in the clinical condition of patients. The evidence accumulated so far indicates that DiAcSpm and DiAcSpd are promising novel tumor markers. They deserve more intensive studies, including studies of their biochemistry and metabolism.     

Oncofetal antigens as tumor markers in the cytologic diagnosis of effusions

To test the value of oncofetal antigens in the cytologic diagnosis of effusions, immunoperoxidase staining with antisera to carcinoembryonic antigen (CEA), alpha fetoprotein (AFP), pregnancy-specific beta 1-glycoprotein (SP1) and placental alkaline phosphatase (PLAP) was carried out on pleural and peritoneal fluids from 72 cases. Sections of formalin-fixed, paraffin-embedded cell blocks were used in most cases; cytocentrifuge preparations were used in some. Reactions were negative with all antisera in 23 of 24 nonmalignant effusions as well as in all 7 cases of malignant mesothelioma and 4 cases of malignant lymphoma. In 24 of 36 confirmed carcinomatous effusions, staining was positive with one or more antisera, including anti-CEA positivity in 23 of the 24 cases. In 5 of the 24 cases with positive staining, a confident diagnosis of malignancy had not been made on routine cytologic preparations. Immunoperoxidase staining for CEA appears to be of supportive value in the cytologic diagnosis of malignancy in effusions.     

Screening with tumor markers

Reviewing the literature it would appear that tumor markers have often flattered to deceive. Early promise does not often seem to be borne out in extended trials. Despite apparently high specificity, very few markers are capable of assisting in a screening process. This brief review attempts to put the roles of tumor markers in perspective and explain how their misapplication has led to misunderstanding of their potential value in a clinical context. It also considers the theoretical basis for their use and highlights how misunderstanding of these can lead to flawed studies and application.     

Peritoneal cytokines as early markers of peritonitis following surgery for colorectal carcinoma: a prospective study

This study was to investigate if measurement of peritoneal cytokines is valuable for an early diagnosis of peritonitis following colorectal surgery. One hundred consecutive patients who were to undergo elective resection for carcinoma of the sigmoid colon or the rectum were investigated. Abdominal exudate was obtained from a drainage tube daily after surgery for measuring interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α. The relationship between peritoneal cytokine levels during the first 3 days after surgery and the development of peritonitis was investigated. Eight patients developed postoperative peritonitis due to anastomotic leakage and pelvic abscess, which was diagnosed on postoperative days 5-8. Peritoneal cytokine levels on postoperative days 1 and 2 were not significantly different between the 8 patients who developed peritonitis and 92 patients who did not: day 1, IL-1βP=0.32, IL-6 P=0.45, TNF-αP=0.85; day 2, IL-1βP=0.26, IL-6 P=0.68, TNF-αP=0.22. In contrast, the cytokine levels on day 3 were significantly higher in patients who developed peritonitis as compared with patients who did not: IL-1βP=0.008, IL-6 P<0.0001, TNF-αP=0.0001. The cytokines significantly increased during the first 3 days in patients who developed peritonitis: IL-1βP=0.049, IL-6 P=0.03, TNF-αP=0.01, while significantly decreased in patients who did not: IL-1βP<0.0001, IL-6 P<0.0001, TNF-αP<0.0001. The outcomes of this investigation showed that the rise in peritoneal IL-1β, IL-6 and TNF-α levels may be an additional early diagnostic predictor of intraabdominal complications following colorectal surgery.     

Cancer cell-secreted proteomes as a basis for searching potential tumor markers: nasopharyngeal carcinoma as a model

Nasopharyngeal carcinoma (NPC) is commonly diagnosed late due to its deep location and vague symptoms. To identify biomarkers for early NPC diagnosis, secreted proteomes of two NPC cell lines were analyzed. Proteins in the NPC cell-line cultured media were systematically identified by SDS-PAGE combined with MALDI-TOF MS. Twenty-three proteins were found in cultured media from both NPC cell lines. Among them, fibronectin, Mac-2 binding protein (Mac-2 BP), and plasminogen activator inhibitor 1 (PAI-1) were further confirmed by Western blot analysis. These three proteins were highly expressed in NPC biopsies, but weakly or not expressed in normal nasopharyngeal tissues. The serum levels of the three proteins were significantly higher in NPC patients (n = 46) than in normal controls (n = 47) (p < 0.01). NPC nude mice model (n = 9) also showed elevated levels of serum Mac-2 BP and PAI-1 compared with tumor-free mice (n = 9) (p < 0.01). Systematic analysis of cancer cell-secreted proteomes combined with animal tumor models can be a feasible, convenient strategy for searching multiple potential tumor markers. Furthermore, our work shows that fibronectin, Mac-2 BP, and PAI-1 may be potential markers for diagnosis of NPC.     

Paradoxical effects of cytokines in tumor immune surveillance and tumor immune escape

The role of cytokines in modulating the formation of new tumors is mediated by their ability to regulate antigen-specific anti-tumor responses and by the activation of non-specific mechanisms, including those involved in the processes of inflammation and innate resistance. Cytokines may influence the growth of tumors by acting directly on tumor cells as growth promoting or growth inhibiting factors or indirectly by attracting inflammatory cell types and affecting angiogenesis. Due to the potency and complexity of cytokine activity against tumor growth, the improvement of cloning techniques and the availability of recombinant forms of different cytokines, a great effort has been made in the recent years to exploit this anti-tumor potential for cancer therapy. This important goal has been difficult to achieve in most cases due to toxicity of most cytokines which could not be dissociated from their anti-tumoral functions. Nevertheless, if well designed, treatment protocols and/or modifications of the cytokine molecules may in some situations augment the anti-tumor effects while limiting the toxicity. One of these molecular approaches could be the design of peptides containing the functional domain of certain cytokines, exemplified by IT9302, a peptide homologous to the functional domain of IL-10, which has demonstrated to increase tumor NK cell sensitivity.     

Glycosaminoglycan-binding properties and secondary structure of the C-terminus of netrin-1

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the "deleted in colorectal carcinoma" gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an alpha-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (K(D) = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, K(D) = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an alpha-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.     

p53 mutations as tumor markers in fine needle aspirates of palpable breast masses

OBJECTIVE: Mutations in p53 exons 5-8 are found in 40-50% of breast carcinomas. We performed a retrospective analysis of p53 mutations in fluid-based, archival fine needle aspirates (FNAs) of breast masses to determine their potential diagnostic utility as breast tumor cell markers. STUDY DESIGN: Residual, fluid-based, archival FNAs of 27 breast masses were retrospectively evaluated by polymerase chain reaction (PCR), single-strand conformational polymorphism analysis (SSCP) and sequencing for p53 exons 5-8. Results were compared with the morphologic diagnoses and genotyping of available excisional biopsy tissue. RESULTS: Six of the twenty-seven cases were found to have a clonal mutation in p53; all six mutated cases showed carcinoma on subsequent excisional biopsy. Definitive cytologic diagnosis of cancer had been possible in only four of the six cases. Identical mutations were found in the excised carcinomas in the five cases with available tissue. None of the 14 aspirates with benign cytology had detectable mutations in p53. CONCLUSION: p53 Mutational analysis by PCR/SSCP/sequencing deserves to be critically studied as a diagnostic criterion in patients with indeterminate or suspicious cytology. Validation studies should be performed to test p53 mutations as molecular diagnostic markers in breast cytology specimens.     

Identification of canine circulating miRNAs as tumor biospecific markers using Next-Generation Sequencing and Q-RT-PCR

Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, ?1306, ?345, ?132, and ?26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future.     

Multiple Glycosaminoglycan-binding Epitopes of Monocyte Chemoattractant Protein-3/CCL7 Enable It to Function as a Non-oligomerizing Chemokine

The interaction of chemokines with glycosaminoglycans (GAGs) facilitates the formation of localized chemokine gradients that provide directional signals for migrating cells. In this study, we set out to understand the structural basis and impact of the differing oligomerization propensities of the chemokines monocyte chemoattractant protein (MCP)-1/CCL2 and MCP-3/CCL7 on their ability to bind GAGs. These chemokines provide a unique comparison set because CCL2 oligomerizes and oligomerization is required for its full in vivo activity, whereas CCL7 functions as a monomer. To identify the GAG-binding determinants of CCL7, an unbiased hydroxyl radical footprinting approach was employed, followed by a focused mutagenesis study. Compared with the size of the previously defined GAG-binding epitope of CCL2, CCL7 has a larger binding site, consisting of multiple epitopes distributed along its surface. Furthermore, surface plasmon resonance (SPR) studies indicate that CCL7 is able to bind GAGs with an affinity similar to CCL2 but higher than the non-oligomerizing variant, CCL2(P8A), suggesting that, in contrast to CCL2, the large cluster of GAG-binding residues in CCL7 renders oligomerization unnecessary for high affinity binding. However, the affinity of CCL7 is more sensitive than CCL2 to the density of heparan sulfate on the SPR surfaces; this is likely due to the inability of CCL7 to oligomerize because CCL2(P8A) also binds significantly less tightly to low than high density heparan sulfate surfaces compared with CCL2. Together, the data suggest that CCL7 and CCL2 are non-redundant chemokines and that GAG chain density may provide a mechanism for regulating the accumulation of chemokines on cell surfaces.     

Clinical significance of blood-based miRNAs as diagnostic and prognostic nucleic acid markers in breast cancer: Comparative to conventional tumor markers

microRNAs (miRNAs) are implicated in carcinogenesis and their expression in biological fluids offer great potential as nucleic acid markers for cancer detection and progression. Authors investigated the expression level of miRNAs (miRNA-21, miRNA-126, and miRNA-155) to evaluate their role as diagnostic and prognostic markers for breast cancer compared with other commonly used protein-based markers (CEA and CA15-3). Serum samples from patients with breast cancer (n = 96), patients with benign breast lesion (n = 47), and healthy individuals (n = 39) were enrolled for detection of miRNA expression levels and protein-based tumor markers using fluorescent real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Correlation among investigated markers with clinicopathological factors and clinical outcomes were determined. Expression of miRNA-21 and miRNA-155 revealed significant increases in patients with breast cancer compared with both benign and control groups, the same result was reported for tumor markers; on the other hand, miRNA-126 was significantly decreased in breast cancer group as compared with the other two groups. miRNA frequencies were significantly related to clinical staging and histological grading as compared with tumor markers. Patients with breast cancer with increased miRNA-21 and miRNA-155 and decreased miRNA-126 expressions had significantly worse disease-free survival, while only miRNA-21 and miRNA-126 showed poor OS (P< 0.005). In conclusion, investigated miRNAs were superior over tumor markers for the early stage of breast cancer especially those with high-risk factor and their assessment in blood facilitates their role as a potential prognostic molecular marker.     

New molecular tumor markers for endometrial cancer

Although the International Federation of Gynecology and Obstetrics officially changed the classification system of endometrial cancer from a clinically staged to a surgically staged disease in 1988, optimal management of patients with endometrial cancer is still controversial. Gynecologists happen to experience that patients with tumors that are identical in grade and stage often have significantly different clinical outcomes or responses to therapy. In order to identify an objective biological factor correlating with tumor aggressiveness, many tumor markers have been investigated. So far, CA125 is one of the most reliable tumor marker for adenocarcinoma of the uterus and frequently used in a clinical setting. Recently, with the advent of molecular biological techniques, many genes and regions of the genome related to endometrial cancer have been identified. We undertook a genome-wide screening to detect genetic changes by comparative genomic hybridization (CGH) in primary endometrioid cancers, since CGH analysis provides comprehensive information concerning relative chromosomal losses and gains in tumors by a single hybridization. In this paper, the usefulness of serum tumor markers and the new promising molecular tumor markers for endometrial cancer are discussed.