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Tumor marker-encoding genes: a bunch of mysterious diamonds in the pile of evolutionary compost

TUMOR MARKERS. . . Tumor markers are proteins or mRNAsthat are detected in higher-than-normal amounts in the blood, urine or body tissues of some patients with certain types of cancer. . Most tumor markers are produced by the tumor itself, but not by normal tissue. Tumor markers could be imm

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Tumor marker-encoding genes: a bunch of mysterious diamonds in the pile of evolutionary compost

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    1. Tumor marker-encoding genes: a bunch of mysterious diamonds in the pile of evolutionary compost

    2. TUMOR MARKERS

    3. Changes in tumor marker concentration during the course of disease:

    4. Known examples of tumor markers

    5. Level of single tumor marker often is not definitive enough

    6. Combining of known tumor markers into marker panels will help to increase sensitivity and specificity of diagnostics

    7. Protein based methods of tumor marker discovery Occasional findings in course of other research Various modifications of SEREX (serological expression of cDNA expression libraries Proteomics–based methods, including: 2D –PAGE analysis SELDI-TOF mass-spectroscopy analysis Reverse proteomics arrays

    14. Surface-enhanced laser desorption/ionization (SELDI-TOF) mass-spectrometry

    16. DNA-based Differential Display

    17. It’s time for computational genomics

    18. Example of ESTs that belong to the same gene/ UNIGENE cluster

    19. Manual verification of cDNA libraries sources

    20. HSAnalyst and ClustOut software

    21. First round of work (year 2000). Cluster-sorting software HSAnalyst

    22. Statistics

    27. Cytokine production and cytotoxic activity of CTLs specific for three Brachyury-derived peptides. A, CD8+ Tcells generated from PBMC of a healthy donor against peptidesT-p2 and T-p3 were stimulated for 24 h in the presence of Brachyury (T)-specific peptides or irrelevant peptide-pulsed autologous DCs. IFN-g was evaluated in the supernatants by ELISA.

    28. Cytotoxic activity of Brachyury-specific CTLs against tumor targets. CTLs established from the blood of (C) a colorectal cancer patient (patient1) and (D) an ovarian cancer patient (patient 2) were used after three IVS for cytotoxic killing of H441 and AsPC-1 tumor cells.

    29. Tumor marker properties of Brachiury were predicted computationally It was the first study to show that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell–mediated cancer immunotherapy. up-regulation of Brachyury occurs in certain tumor tissues and cancer cell lines Brachyury-specific CTLs can be generated from the blood of cancer patients and normal donors, which, in turn, can effectively lyse Brachyury-expressing tumor cells.

    30. Recent round of work (year 2006). Cluster-sorting software ClustOut

    31. Tumor prevalent clusters

    32. Known tumor markers in cancer-specific subtraction

    33. Prospective marker proteins in cancer-specific subtraction

    34. Known human genes represented by only tumor-derived EST

    35. Reverse subtraction: genes prevalent in normal samples

    36. Normal prevalent clusters

    37. Non-coding RNAs in tumor-specific and normal–specific subtractions In normal – prevalent clusters: 399 out of 1048 (36,9%) In tumor prevalent clusters: 466 out of 501 (87%)

    38. Possible explanation: Tumors express more non-coding mRNA than normal tissue due to: -- overall weakness of gene expression control (probably due to demethylation of genome) Awakening of dormant promoters

    39. Experimental study of Tumor Specificity: Whether software based predictions are true or not??? Kozlov et al., 2003 Krukovskaja et al., 2004

    50. Three other normal panels looks same as previous; Conclusion: Hs.154173 is a Tumor-Specific Sequence

    55. Back to fundamental aspect of the tumor-specific mRNAs

    56. Closer look into well-established tumor-markers 1) For most of the known tumor markers no clear function in the adult human cells is shown; 2) Looks like these genes not expressed in normal tissues just because they not needed there; 3) Looks like these genes are expressed in tumors just because tumors don’t care !!!

    57. VISTA – special visualization tool for distant cross-species comparison

    58. How normal gene looks in VISTA

    59. How tumor marker gene looks in VISTA (MAGE A4)

    60. You don’t believe me? Here is MAGE A9 – another one.

    61. More detailed VISTA view

    62. More detailed VISTA view

    63. Some genes are right in the middle: some exons are conservative, some are not (non-coding ones)

    64. Graphical alignments

    65. What about our own flock of predicted tumor markers ???

    67. Evolution often goes by duplications

    68. 22 of 170 cancer specific gene clusters are recently (only in primates) duplicated in human genome !!!

    69. IDEA: In course of evolution new sequences become ready to be expressed as they got joined to dormant promoter But unnecessary promotors are thoroughly suppressed in well-differentiated adult cells In tumors all these “yet-to-be” genes got their chance

    70. Same is true for “parts of the genes” – separate exons, especially non-coding exons

    71. Something to think about: 1. Can we use these non-coding tumor-specific mRNA as a tumor markers and targets for immunotherapy? (protein-coding genes could be used more readily) Yes. They always encode short ORFs linked with AUGs. Such short ORFs are TRUE non-self antigens.

    72. 2. Why known tumor markers have carcino-embryonic or carcino-testicular profiles of expression? -- embryos contain lots of less differentiated cells with relaxed expression control; -- testes are covered by blood-testes barrier, so immune system is not watching for an expression of non-self ORFs It explains broadest pattern of expression observed in tested Something to think about:

    73. George Mason University, Fairfax, VA, USA A.Baranova, M.Sikaroodi, G,Manyam, P. Gillevet

    74. The Biomedical Center, St.Petersburg, Russia A.P. Kozlov, L.L.Krukovskaja, D.Polev, I. Duhovlinov, Yu.Galachjants, N.Samusik

    75. Vavilov Institute of General Genetics, Moscow, Russia T.Tyazhelova NCI, NIH C.Palena, J.Schlom S.O’Brien

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