Patent Application: US-95389907-A

Abstract:
the present application relates to gene analysis and , in particular , to gene expression profiling for identifying molecular signature of cancer disease , in particular the g1 phase of the cell cycle , such as myeloproliferative disorders or breast cancer and studying cancer . more particularly , the application is directed to a method for analyzing differential gene expression associated with cancer disease , in particular the g1 phase of the cell cycle , such as myeloproliferative disorders or breast cancer comprising detection of upregulation and / or downregulation of a pool of polynucleotide sequences in a cell or tissue sample , said pool corresponding to all or part the polynucleotide sequences , subsequences or complements thereof , of the genes listed in tables 1 , 2 and 3 .

Description:
myeloproliferative disorders ( mpd ) are clonal hematopoietic diseases characterized by the proliferation and expansion of one or several myeloid cell lineages in the bone marrow . during the chronic phase , the cells follow their normal differentiation pathway and become mature blood cells . during a second phase , an acute syndrome may occur . the conventional classification separates mpds in clinical entities . these include chronic myeloid leukemia ( cml ), chronic neutrophilic leukemia , chronic eosinophilic leukemia , polycythemia vera ( pv ), essential thrombocythemia ( et ) and idiopathic myelofibrosis ( imf ). mpds with different syndromes and molecular abnormalities are grouped in non - classical mpds . systemic mastocytosis is not classically included in mpds but is a related disease . we studied the gene expression profiles of murine ba / f3 cells transfected by various oncogenic mpd kinases by using whole - genome affymetrix 430 2 . 0 mouse oligonucleotide microarrays ( www . affymetrix . com ). ba / f3 cells were grown in rpmi medium supplemented with 10 % fetal calf serum ( fcs ) and il3 . expression of an mpd kinase bypasses the il3 dependence of ba / f3 cells . rna was extracted by using trizol ( trizol reagent , invitrogen life technologies , carlsbad , calif .) from frozen pellets of : i )— ba / f3 cells , ii )— ba / f3 transfected with different pcdna expression vectors expressing a mutant , kinase - defective fop - fgfr1 kd or an oncogenic mpd kinase ( bcr - abl , fop - fgfr1 , cep1 - fgfr1 , bcr - fgfr1 ), and iii )— ba / f3 transfected by the migr vector , either empty ( migr ) or with jak2 ( jak2 wt , mutated v617f jak2 or mutated v617f ind jak2 i . e . able to grow independently of il3 ). before rna extraction , cells were starved for 7 hours in rpmi plus 0 . 5 % fcs . rna integrity was controlled by microanalysis ( agilent bioanalyzer , palo alto , calif .). preparation of crna , hybridizations , washes , detection and quantification were done as recommended by the supplier ( affymetrix ). data were analyzed by the rma ( robust multichip average ) method in r using bioconductor and associated package . before analysis , a filtering process removed from the dataset the genes with low and poorly measured expression as defined by an expression value inferior to 100 units in all samples , retaining 17 . 885 genes / ests . for paired samples , rna was prepared independently from different cultures of cells . the correlation between paired samples ranged between 0 . 97 and 0 . 98 . gene expression profiles of ba / f3 cells transfected by fusion or mutated kinases ( 9 samples : bcr - abl , 2 bcr - fgfr1 , 2 cep1 - fgfr1 , 2 fop - fgfr1 , 2 v617f jak2 ) were compared to that of control cells ( 6 samples ) including parental ba / f3 cells ( 4 samples ) and ba / f3 expressing a kinase - defective mutant of fop - fgfr1 ( 2 samples ). supervised analysis , based on 17 , 885 filtered probe - sets , identified 294 differentially expressed probe sets ( theoretical number of produced false positives = 1 . 7 ) ( fig1 a , b ), representing 228 genes and 8 ests , of which 188 were upregulated and 48 downregulated in activated kinase - expressing cells ( tables 2 and 3 ). to translate the rna expression profiles into functionality , discriminator genes / ests were interrogated by onto - express . 7 table 1 represents the most significant ( p - value inferior at 3 · 10 − 2 ) and most often represented ( including at least 3 genes ) biological processes . many of the upregulated genes encode nucleolar proteins involved in “ ribosome biogenesis ” ( go : 0007046 , 6 genes , p = 4 . 28 · 10 − 11 ), “ rrna processing ” ( go : 0006364 ; 7 genes , p = 3 . 07 · 10 − 11 ), and “ protein biosynthesis ” ( go : 0006412 , 9 genes , p = 3 . 60 · 10 − 05 ). upregulated genes encode nucleolar proteins ( cirh1a , larp1 , nol1 , nol11 , nol5 , nol5a , nola1 , nola2 , nolc1 , mki67ip , sfrs2 , surf6 ), ribosomal proteins ( rpl3 , rpl12 , rpl41 , rps9 , rrs1 ), small nuclear ribonucleoproteins and interactors ( u3 / mphosph10 , lsm2 , rnu22 , rnu3ip2 ), components of rna polymerase i ( polr1a , polr1b ), ii ( polr2h , taf9 ) and iii ( polr3e , polr3h ), dead - box ( ddx18 , ddx56 ) and wd repeat ( wdr4 , wdr43 , wdr74 , wdr77 , grwd1 , pwp1 ) proteins , eukaryotic initiation and elongation factors ( eif1a , eif3s1 , eif3s4 , eef1e1 ), and components of the exosome ( exosc1 , exosc2 , exosc6 ). upregulated genes also encode proteins of the nol5a - associated preribosomal ribonucleoprotein complex involved in pre - rrna processing : nol5a , ppan , nolc1 , and bxdc2 . the gene encoding ebna1bp2 was upregulated ; it encodes a protein that binds to nucleolar fgf3 and is regularly upregulated in tumors . the most upregulated sequence was gas5 , a non - protein - coding multiple small nucleolar rna ( snorna ). other significant processes included “ protein folding ” ( go : 0006457 ; 4 genes , p = 6 . 72 · 10 − 03 ), “ ubiquitin - dependent protein catabolism ” ( go : 0006511 , 3 genes , p = 1 . 47 · 10 − 02 ), “ nuclear mrna splicing , via spliceosome ” ( go : 0000398 , 3 genes , p = 1 . 65 · 10 − 02 ), and “ regulation of cell cycle ” ( go : 0000074 , 3 genes , p = 1 . 65 · 10 − 02 ). the second major category of upregulated genes encode ccnd2 ( cyclin d2 ) and cdc25a , two major regulators needed for g1 progression . ccnd2 rna was found upregulated by bcr - abl in previous gene expression studies . cyclin d2 is necessary for bcr - abl - induced activity . inhibition of v617f jak2 correlates with decreased expression of cyclin d2 . other g1 cyclins may play a role in the oncogenic activity of fusion kinases but cyclin d2 seems to be a rate - limiting element . we used western blot analysis to validate the differential expression of cyclin d2 . the amount of cyclin d2 protein was increased in ba / f3 cells expressing activated kinases as compared to controls ( fig1 c ), in agreement with mrna expression results . myc directly or indirectly regulates the g1 phase of the cell cycle . the list of upregulated genes included myc . many genes upregulated by myc and nmyc oncogenes were also upregulated in our experiments , including ccnd2 , cdc25a and others ( ddx18 , ebna1bp2 , eef1e1 , mat2a , mki67ip , nol5a , nola1 , phb , sfrs2 , shmt1 , slc16a1 , surf6 , srm , rpl3 , rpl12 , rpl41 , rps9 and rrs1 ). this similarity suggests that myc proteins and mpd kinases have similar oncogenic effects , whose main target would be the cdkn2 - rb protein pathway during the g1 phase of the cell cycle . once induced , myc may in turn act on the transcription of g1 / s regulators and genes involved in protein synthesis . mpd fusion kinases are thought to target the hematopoietic stem cell . activation of myc is in perfect agreement with what we know of stem cell proliferation . a similar program was also turned on by il3 stimulation ( not shown ). downregulated genes were more difficult to classify with onto - express , but several encode proteins with known or potential inhibitory function such as pias3 , an inhibitor of stat3 , one of the main substrates of mpd kinases , and regulator of cdc25a , erbin , and plzf / zbtb16 , a myc repressor . we tested the validity of our classification by the “ leave - one - out ” cross - validation method . iteratively , one of the 15 samples was removed , and a multigene predictor was generated from the remaining samples : 93 % of samples were correctly assigned by the predictors with a sensitivity of 89 % and a specificity of 100 %. thus , in ba / f3 cells , mpd fusion kinases induce both g1 activators and protein synthesis components , thus starting the cell proliferation machinery . this effect may be mediated by the pi3 kinase - akt - tor pathway , which controls and coordinates both protein synthesis and early phases of the cell cycle . prominent downstream targets of the akt pathway are cyclins d1 , d2 and myc . the subject matter of the references set forth below are hereby incorporated by reference in their entirety : 1 . percy m j , mcmullin m f . the v617f jak2 mutation and the myeloproliferative disorders . hematol oncol 2005 ; 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