Patent Document

[0001]    This application was made using funds provided by the United States government under grant nos. NIH-CA 62924 and NIH-CA 43460. The United States government therefore retains certain rights in the invention. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The invention relates to the fields of diagnostic tests and therapeutic methods for cancer. 
       BACKGROUND OF THE INVENTION 
       [0003]    PI3Ks are lipid kinases that function as signal transducers downstream of cell surface receptors and mediate pathways important for cell growth, proliferation, adhesion, survival and motility (1, 2). Although increased PI3K activity has been observed in many colorectal and other tumors (3, 4), no intragenic mutations of PI3K have been identified. 
         [0004]    Members of the PIK3 pathway have been previously reported to be altered in cancers, for example, the PTEN tumor suppressor gene (15, 16), whose function is to reverse the phosphorylation mediated by PI3Ks (17, 18). Reduplication or amplification of the chromosomal regions containing PIK3CA and AKT2 has been reported in some human cancers (2, 19, 20), but the genes that are the targets of such large-scale genetic events have not been and cannot easily be defined. 
       BRIEF SUMMARY OF THE INVENTION 
       [0005]    In a first embodiment a method is provided for assessing cancer in a human tissue suspected of being cancerous of a patient. A non-synonymous, intragenic mutation in a PIK3CA coding sequence is detected in a body sample of a human suspected of having a cancer. The human is identified as likely to have a cancer if a non-synonymous, intragenic mutation in PIK3CA coding sequence is determined in the body sample. 
         [0006]    In a second embodiment of the invention a method is provided for inhibiting progression of a tumor in a human. An antisense oligonucleotide or antisense construct is administered to a tumor. The antisense oligonucleotide or RNA transcribed from the antisense construct is complementary to mRNA transcribed from PIK3CA. The amount of p110α protein expressed by the tumor is thereby reduced. 
         [0007]    Another embodiment of the invention provides a method of inhibiting progression of a tumor in a human. siRNA comprising 19 to 21 bp duplexes of a human PIK3CA mRNA with 2 nt 3′ overhangs are administered to the human. One strand of the duplex comprises a contiguous sequence selected from mRNA transcribed from PIK3CA (SEQ ID NO: 2). The amount of p110α protein expressed by the tumor is thereby reduced. 
         [0008]    According to another aspect of the invention a method is provided for inhibiting progression of a tumor. A molecule comprising an antibody binding region is administered to a tumor. The antibody binding region specifically binds to PIK3CA (SEQ ID NO: 3). 
         [0009]    Another embodiment of the invention provides a method of identifying candidate chemotherapeutic agents. A wild-type or activated mutant p110α (SEQ ID NO: 3) is contacted with a test compound. p110α activity is then measured. A test compound is identified as a candidate chemotherapeutic agent if it inhibits p110α activity. 
         [0010]    Still another embodiment of the invention is a method for delivering an appropriate chemotherapeutic drug to a patient in need thereof. A non-synonymous, intragenic mutation in a PIK3CA coding sequence (SEQ ID NO: 1) is determined in a test tissue of a patient. A p110α inhibitor is administered to the patient. 
         [0011]    An additional aspect of the invention provides a set of one or more primers for amplifying and/or sequencing PIK3CA. The primers are selected from the group consisting of forward primers, reverse primers and sequencing primers. The forward primers are selected from the group consisting of: SEQ ID NO: 6 to 158; the reverse primers are selected from the group consisting of: SEQ ID NO: 159 to 310; and the sequencing primers are selected from the group consisting of: SEQ ID NO: 311 to 461. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0012]      FIG. 1 . Detection of mutations in of PIK3CA. Representative examples of mutations in exons 9 and 20. In each case, the top sequence chromatogram was obtained from normal tissue and the three lower sequence chromatograms from the indicated tumors. Arrows indicate the location of missense mutations. The nucleotide and amino acid alterations are indicated above the arrow. 
           [0013]      FIG. 2 . Distribution of mutations in PIK3CA. Arrows indicate the location of missense mutations, and boxes represent functional domains (p85BD, p85 binding domain; RBD, Ras binding domain; C2 domain; Helical domain; Kinase domain). The percentage of mutations detected within each region in cancers is indicated below. 
           [0014]      FIGS. 3A-3C . Increased lipid kinase activity of mutant p110α. NIH3T3 cells were transfected with empty vector or with vector constructs containing either wild-type p110α or mutant p110α (H1047R) as indicated above the lanes. Immunoprecipitations were performed either with control IgG or anti-p85 polyclonal antibodies. ( FIG. 3A ) Half of the immunoprecipitates were subjected to a PI3-kinase assay using phosphatidylinositol as a substrate. “PI3P” indicates the position of PI-3-phosphate determined with standard phosphatidyl markers and “Ori” indicates the origin. ( FIG. 3B ) The other half of the immunoprecipitates was analyzed by western blotting with anti-p110α antibody. ( FIG. 3C ) Cell lysates from transfected cells contained similar amounts of total protein as determined by western blotting using an anti-α-tubulin antibody. Identical results to those shown in this figure were observed in three independent transfection experiments. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0015]    The clustering of mutations within PIK3CA make it an excellent marker for early detection or for following disease progression. Testing focused in the clustered regions will yield most of the mutant alleles. 
         [0016]    The human PIK3CA coding sequence is reported in the literature and is shown in SEQ ID NO: 1. This is the sequence of one particular individual in the population of humans. Humans vary from one to another in their gene sequences. These variations are very minimal, sometimes occurring at a frequency of about 1 to 10 nucleotides per gene. Different forms of any particular gene exist within the human population. These different forms are called allelic variants. Allelic variants often do not change the amino acid sequence of the encoded protein; such variants are termed synonymous. Even if they do change the encoded amino acid (non-synonymous), the function of the protein is not typically affected. Such changes are evolutionarily or functionally neutral. When human PIK3CA is referred to in the present application all allelic variants are intended to be encompassed by the term. The sequence of SEQ ID NO: 1 is provided merely as a representative example of a wild-type human sequence. The invention is not limited to this single allelic form of PIK3CA. For purposes of determining a mutation, PIK3CA sequences determined in a test sample can be compared to a sequence determined in a different tissue of the human. A difference in the sequence in the two tissues indicates a somatic mutation. Alternatively, the sequence determined in a PIK3CA gene in a test sample can be compared to the sequence of SEQ ID NO: 1. A difference between the test sample sequence and SEQ ID NO: 1 can be identified as a mutation. Tissues suspected of being cancerous can be tested, as can body samples that may be expected to contain sloughed-off cells from tumors or cells of cancers. Suitable body samples for testing include blood, serum, plasma, sputum, urine, stool, nipple aspirate, saliva, and cerebrospinal fluid. 
         [0017]    Mutations in PIK3CA cluster in exons 9 (SEQ ID NO: 4) and 20 (SEQ ID NO: 5). Other mutations occur, but these two exons appear to be the hotspots for mutations. Many mutations occur in PIK3CA&#39;s helical domain (nt 1567-2124 of SEQ ID NO: 2) and in its kinase domain (nt 2095-3096 of SEQ ID NO: 2). Fewer occur in PIK3CA&#39;s P85BD domain (nt 103-335 of SEQ ID NO: 2). Mutations have been found in exons 1, 2, 4, 5, 7, 9, 13, 18, and 20. Any combination of these exons can be tested, optionally in conjunction with testing other exons. Testing for mutations can be done along the whole coding sequence or can be focused in the areas where mutations have been found to cluster. Particular hotspots of mutations occur at nucleotide positions 1624, 1633, 1636, and 3140 of PIK3CA coding sequence. 
         [0018]    PIK3CA mutations have been found in a variety of different types of tumors. Thus any of a variety of tumors can be tested for PIK3CA mutations. These tissues include, without limitation: colorectal tissue, brain tissue, gastric tissue, breast tissue, and lung tissue. 
         [0019]    Any type of intragenic mutation can be detected. These include substitution mutations, deletion mutations, and insertion mutations. The size of the mutations is likely to be small, on the order of from 1 to 3 nucleotides. Mutations which can be detected include, but are not limited to G1624A, G1633A, C1636A, A3140G, G113A, T1258C, G3129T, C3139T, and G2702T. Any combination of these mutations can be tested. 
         [0020]    The mutations that are found in PIK3CA appear to be activating mutations. Thus therapeutic regimens involving inhibition of p110α activity or expression can be used to inhibit progression of a tumor in a human. Inhibitory molecules which can be used include antisense oligonucleotides or antisense constructs, a molecule comprising an antibody binding region, and siRNA molecules. Molecules comprising an antibody binding region can be full antibodies, single chain variable regions, antibody fragments, antibody conjugates, etc. The antibody binding regions may but need not bind to epitopes contained within the kinase domain (nt 2095-3096 of SEQ ID NO: 2) of PIK3CA, the helical domain (nt 1567-2124 of SEQ ID NO: 2) of PIK3CA, or the P85BD domain (nt 103-335 of SEQ ID NO: 2) of PIK3CA. 
         [0021]    Antisense constructs, antisense oligonucleotides, RNA interference constructs or siRNA duplex RNA molecules can be used to interfere with expression of PIK3CA. Typically at least 15, 17, 19, or 21 nucleotides of the complement of PIK3CA mRNA sequence are sufficient for an antisense molecule. Typically at least 19, 21, 22, or 23 nucleotides of PIK3CA are sufficient for an RNA interference molecule. Preferably an RNA interference molecule will have a 2 nucleotide 3′ overhang. If the RNA interference molecule is expressed in a cell from a construct, for example from a hairpin molecule or from an inverted repeat of the desired PIK3CA sequence, then the endogenous cellular machinery will create the overhangs. siRNA molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, or other methods known in the art. See Hannon, G J, 2002, RNA Interference,  Nature  418: 244-251; Bernstein E et al., 2002, The rest is silence.  RNA  7: 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double-strand.  Curr. Opin. Genetics  &amp;  Development  12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells.  Science  296: 550-553; Lee N S, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.  Nature Biotechnol.  20:500-505; Miyagishi M, and Taira K. (2002). U6-promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells.  Nature Biotechnol.  20:497-500; Paddison P J, Caudy A A, Bernstein E, Hannon G J, and Conklin D S. (2002). Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.  Genes  &amp;  Dev.  16:948-958; Paul C P, Good P D, Winer I, and Engelke D R. (2002). Effective expression of small interfering RNA in human cells.  Nature Biotechnol.  20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester W C, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.  Proc. Natl. Acad. Sci. USA  99(6):5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells.  Proc. Natl. Acad. Sci. USA  99(9):6047-6052. 
         [0022]    Antisense or RNA interference molecules can be delivered in vitro to cells or in vivo, e.g., to tumors of a mammal. Typical delivery means known in the art can be used. For example, delivery to a tumor can be accomplished by intratumoral injections. Other modes of delivery can be used without limitation, including: intravenous, intramuscular, intraperitoneal, intraarterial, local delivery during surgery, endoscopic, subcutaneous, and per os. In a mouse model, the antisense or RNA interference can be adminstered to a tumor cell in vitro, and the tumor cell can be subsequently administered to a mouse. Vectors can be selected for desirable properties for any particular application. Vectors can be viral or plasmid. Adenoviral vectors are useful in this regard. Tissue-specific, cell-type specific, or otherwise regulatable promoters can be used to control the transcription of the inhibitory polynucleotide molecules. Non-viral carriers such as liposomes or nanospheres can also be used. 
         [0023]    Using the p110α protein according to the invention, one of ordinary skill in the art can readily generate antibodies which specifically bind to the proteins. Such antibodies can be monoclonal or polyclonal. They can be chimeric, humanized, or totally human. Any functional fragment or derivative of an antibody can be used including Fab, Fab′, Fab2, Fab′2, and single chain variable regions. So long as the fragment or derivative retains specificity of binding for the endothelial marker protein it can be used. Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific. 
         [0024]    Techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used. According to one particularly preferred embodiment, fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody. See for example, Nina D. Russel, Jose R. F. Corvalan, Michael L. Gallo, C. Geoffrey Davis, Liise-Anne Pirofski. Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoglobulin Loci  Infection and Immunity  April 2000, p. 1820-1826; Michael L. Gallo, Vladimir E. Ivanov, Aya Jakobovits, and C. Geoffrey Davis. The human immunoglobulin loci introduced into mice: V (D) and J gene segment usage similar to that of adult humans  European Journal of Immunology  30: 534-540, 2000; Larry L. Green. Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies  Journal of Immunological Methods  231 11-23, 1999; Yang X-D, Corvalan J R F, Wang P, Roy C M-N and Davis C G. Fully Human Anti-interleukin-8 Monoclonal Antibodies: Potential Therapeutics for the Treatment of Inflammatory Disease States.  Journal of Leukocyte Biology  Vol. 66, pp 401-410 (1999); Yang X-D, Jia X-C, Corvalan J R F, Wang P, C G Davis and Jakobovits A. Eradication of Established Tumors by a Fully Human Monoclonal Antibody to the Epidermal Growth Factor Receptor without Concomitant Chemotherapy.  Cancer Research Vol.  59, Number 6, pp1236-1243 (1999); Jakobovits A. Production and selection of antigen-specific fully human monoclonal antibodies from mice engineered with human Ig loci.  Advanced Drug Delivery Reviews  Vol. 31, pp: 33-42 (1998); Green L and Jakobovits A. Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes.  J. Exp. Med. Vol.  188, Number 3, pp: 483-495 (1998); Jakobovits A. The long-awaited magic bullets: therapeutic human monoclonal antibodies from transgenic mice.  Exp. Opin. Invest. Drugs  Vol. 7(4), pp: 607-614 (1998); Tsuda H, Maynard-Currie K, Reid L, Yoshida T, Edamura K, Maeda N, Smithies O, Jakobovits A. Inactivation of Mouse HPRT locus by a 203-bp retrotransposon insertion and a 55-kb gene-targeted deletion: establishment of new HPRT-Deficient mouse embryonic stem cell lines.  Genomics  Vol. 42, pp: 413-421 (1997); Sherman-Gold, R. Monoclonal Antibodies: The Evolution from &#39;80s Magic Bullets To Mature, Mainstream Applications as Clinical Therapeutics.  Genetic Engineering  News Vol. 17, Number 14 (August 1997); Mendez M, Green L, Corvalan J, Jia X-C, Maynard-Currie C, Yang X-d, Gallo M, Louie D, Lee D, Erickson K, Luna J, Roy C, Abderrahim H, Kirschenbaum F, Noguchi M, Smith D, Fukushima A, Hales J, Finer M, Davis C, Zsebo K, Jakobovits A. Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice.  Nature Genetics Vol.  15, pp: 146-156 (1997); Jakobovits A. Mice engineered with human immunoglobulin YACs: A new technology for production of fully human antibodies for autoimmunity therapy.  Weir&#39;s Handbook of Experimental Immunology, The Integrated Immune System  Vol. IV, pp: 194.1-194.7 (1996); Jakobovits A. Production of fully human antibodies by transgenic mice.  Current Opinion in Biotechnology  Vol. 6, No. 5, pp: 561-566 (1995); Mendez M, Abderrahim H, Noguchi M, David N, Hardy M, Green L, Tsuda H, Yoast S, Maynard-Currie C, Garza D, Gemmill R, Jakobovits A, Klapholz S. Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells.  Genomics Vol.  26, pp: 294-307 (1995); Jakobovits A. YAC Vectors: Humanizing the mouse genome.  Current Biology  Vol. 4, No. 8, pp: 761-763 (1994); Arbones M, Ord D, Ley K, Ratech H, Maynard-Curry K, Otten G, Capon D, Tedder T. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice.  Immunity  Vol. 1, No. 4, pp: 247-260 (1994); Green L, Hardy M, Maynard-Curry K, Tsuda H, Louie D, Mendez M, Abderrahim H, Noguchi M, Smith D, Zeng Y, et. al. Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs.  Nature Genetics  Vol. 7, No. 1, pp: 13-21 (1994); Jakobovits A, Moore A, Green L, Vergara G, Maynard-Curry K, Austin H, Klapholz S. Germ-line transmission and expression of a human-derived yeast artificial chromosome.  Nature  Vol. 362, No. 6417, pp: 255-258 (1993); Jakobovits A, Vergara G, Kennedy J, Hales J, McGuinness R, Casentini-Borocz D, Brenner D, Otten G. Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production.  Proceedings of the National Academy of Sciences USA  Vol. 90, No. 6, pp: 2551-2555 (1993); Kucherlapati et al., U.S. Pat. No. 6,1075,181. 
         [0025]    Antibodies can also be made using phage display techniques. Such techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Single chain Fv can also be used as is convenient. They can be made from vaccinated transgenic mice, if desired. Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. 
         [0026]    Antibodies can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled antibodies can be used for diagnostic techniques, either in vivo, or in an isolated test sample. Antibodies can also be conjugated, for example, to a pharmaceutical agent, such as chemotherapeutic drug or a toxin. They can be linked to a cytokine, to a ligand, to another antibody. Suitable agents for coupling to antibodies to achieve an anti-tumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF); photosensitizers, for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 ( 131 I), yttrium-90 ( 90 Y), bismuth-212 ( 212 Bi), bismuth-213 ( 213 Bi), technetium-99m ( 99m Tc), rhenium-186 ( 186 Re), and rhenium-188 ( 188 Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinese cobra (naja naja atra), and gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by  Aspergillus restrictus ), saporin (a ribosome inactivating protein from  Saponaria officinalis ), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing antitumor agents (e.g. antisense oligonucleotides, plasmids which encode for toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab). 
         [0027]    Those of skill in the art will readily understand and be able to make such antibody derivatives, as they are well known in the art. The antibodies may be cytotoxic on their own, or they may be used to deliver cytotoxic agents to particular locations in the body. The antibodies can be administered to individuals in need thereof as a form of passive immunization. 
         [0028]    Given the success of small molecule protein kinase inhibitors, one can develop specific or non-specific inhibitors of p110α for treatment of the large number of patients with these mutations or cancers generally. It is clearly possible to develop broad-spectrum PI3K inhibitors, as documented by studies of LY294002 and wortmannin (2, 21, 22). Our data suggest that the development of more specific inhibitors that target p110α but not other PI3Ks would be worthwhile. 
         [0029]    Candidate chemotherapeutic agents can be identified as agents which inhibit p110α activity or expression. Test compounds can be synthetic or naturally occurring. They can be previously identified to have physiological activity or not. Tests on candidate chemotherapeutic agents can be run in cell-free systems or in whole cells. p110α activity can be tested by any means known in the art. These include methods taught in references 2, 22 and in Truitt et al.,  J. Exp. Med.,  179, 1071-1076 (1994). Expression can be monitored by determining PI3KCA protein or mRNA. Antibody methods such as western blotting can be used to determine protein. Northern blotting can be used to measure mRNA. Other methods can be used without limitation. When testing for chemotherapeutic agents, the p110α used in the assay can be a wild-type or an activated form. The activated form may contain a substitution mutation selected from the group consisting of E542K, E545K, Q546K, and H1047R. Moreover, inhibitors can be tested to determine their specificity for either p110α or an activated form of p110α. Comparative tests can be run against similar enzymes including PIK3CB, PIK3CG, PIK3C2A, PIK3C2B, PIK3C2G, PIK3C3, A-TM, ATR, FRAP1, LAT1-3TM, SMG1, PRKDC, and TRRAP to determine the relative specificity for the p110α enzyme. 
         [0030]    Once a non-synonymous, intragenic mutation in a PIK3 CA coding sequence is identified in a test tissue of a patient, that information can be used to make therapeutic decisions. Patients with such mutations are good candidates for therapy with a p110α inhibitor. Such inhibitors can be specific or general for the family of inhibitors. Such inhibitors include LY294002 and wortmannin. Such inhibitors further include molecules comprising an antibody binding region specific for p110α. Such molecules are discussed above. 
         [0031]    Sets of primers for amplifying and/or sequencing PIK3CA can be provided in kits or assembled from components. Useful sets include pairs of forward and reverse primers optionally teamed with sequencing primers. The forward primers are shown in SEQ ID NO: 6 to 158. The reverse primers are shown in SEQ ID NO: 159 to 310. The sequencing primers are shown in: SEQ ID NO: 311 to 461. Pairs or triplets or combinations of these pairs or triplets can be packaged and used together to amplify and/or sequence parts of the PIK3CA gene. Pairs can be packaged in single or divided containers. Instructions for using the primers according to the methods of the present invention can be provided in any medium which is convenient, including paper, electronic, or a world-wide web address. 
         [0032]    While the invention has been described with respect to specific examples including presently preferred modes of carrying out the invention, those skilled in the art will appreciate that there are numerous variations and permutations of the above described systems and techniques that fall within the spirit and scope of the invention as set forth in the appended claims. 
       EXAMPLES 
     Example 1 
     This Example Demonstrates that the PIK3CA Gene is the Predominant Target of Mutations in this Gene Family 
       [0033]    To evaluate whether PI3Ks is genetically implicated in tumorigenesis, we directly examined the DNA sequences of members of this gene family in colorectal cancers. 
         [0034]    PI3K catalytic subunits are divided into three major classes depending on their substrate specificity (5). Additionally, a set of more distantly related proteins, including members of the mTOR family, constitute a fourth class (6). We used Hidden Markov models to identify 15 human genes containing kinase domains related to those of known PI3Ks in the human genome (7). These comprised seven PI3Ks, six members of the mTOR subfamily and two uncharacterized PI3K-like genes (Table 1). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 PI3K genes analyzed 
               
             
          
           
               
                   
                 Celera 
                 Genbank 
                   
                   
               
               
                 Gene name 
                 Accession 
                 Accession 
                 Alternate names 
                 Group* 
               
               
                   
               
               
                 PIK3CA 
                 hCT1640694 
                 NM_006218 
                 p110-alpha 
                 Class IA 
               
               
                 PIK3CB 
                 hCT7084 
                 NM_006219 
                 PIK3C1, p110-beta 
                 Class IA 
               
               
                 PIK3CD 
                 hCT2292011 
                 NM_005026 
                 p110-delta 
                 Class IA 
               
               
                 PIK3CG 
                 hCT7976 
                 NM_002649 
                 PI3CG, PI3K-gamma 
                 Class IB 
               
               
                 PIK3C2A 
                 hCT2270768 
                 NM_002645 
                 CPK, PI3-K-C2A, PI3K-C2alpha 
                 Class II 
               
               
                 PIK3C2B 
                 hCT7448 
                 NM_002646 
                 C2-PI3K, PI3K-C2beta 
                 Class II 
               
               
                 PIK3C2G 
                 hCT1951422 
                 NM_004570 
                 PI3K-C2-gamma 
                 Class II 
               
               
                 PIK3C3 
                 hCT13660 
                 NM_002647 
                 Vps34 
                 Class III 
               
               
                 ATM 
                 hCT29277 
                 NM_000051 
                 AT1, ATA, ATC, ATD, ATE, ATDC 
                 Class IV 
               
               
                 ATR 
                 hCT1951523 
                 NM_001184 
                 FRP1, SCKL, SCKL1 
                 Class IV 
               
               
                 FRAP1 
                 hCT2292935 
                 NM_004958 
                 FRAP, MTOR, FRAP2, RAFT1, RAPT1 
                 Class IV 
               
               
                 SMG1 
                 hCT2273636 
                 NM_014006 
                 ATX, LIP, KIAA0421 
                 Class IV 
               
               
                 PRKDC 
                 hCT2257127 
                 NM_006904 
                 p350, DNAPK, DNPK1, HYRC1, XRCC7 
                 Class IV 
               
               
                 TRRAP 
                 hCT32594 
                 NM_003496 
                 TR-AP, PAF400 
                 Class IV 
               
               
                 none 
                 hCT2257641 
                 none 
                   
                 Class IV 
               
               
                 none 
                 hCT13051 
                 none 
                   
                 Class IV 
               
               
                   
               
               
                 *PI3K genes are grouped into previously described classes (S3, S4). Class I, II and III comprise PI3K catalytic subunits, while class IV comprises PI3K-like genes including members of the mTOR (target of rapamycin), ATM (ataxia telangiectasia mutated), and DNAPK (DNA-dependent protein kinase) subfamilies, as well as two previously uncharacterized genes. 
               
             
          
         
       
     
         [0035]    We initially examined 111 exons encoding the predicted kinase domains of these genes (Table 2). The exons were polymerase chain reaction (PCR) amplified and directly sequenced from genomic DNA of 35 colorectal cancers (8). Only one of the genes (PIK3CA) contained any somatic (i.e., tumor-specific) mutations. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Primers used for PCR amplification and sequencing 
               
             
          
           
               
                 Gene and Exon Name 
                 Forward Primer 1   
                 Reverse primer 2   
                 Sequencing Primer 3   
               
               
                   
               
             
          
           
               
                 hCT2270768-Ex21 
                 TTCCAGCCTGGGTAACAAAG 
                 CGTCAGAACAAGACCCTGTG 
                 AAAGGGGAAATGCGTAGGAC 
                   
               
               
                   
               
               
                 hCT2270768-Ex22 
                 CCTGACCTCAGGTGTTCTGC 
                 CCCGGCCACTAAGTTATTTTTC 
                 TCCCAAAGTGCTGGGATTAC 
               
               
                   
               
               
                 hCT2270768-Ex23 
                 TGCACATTCTGCACGTGTATC 
                 CTGCCATTAAATGCGTCTTG 
                 CCAGAACTTAAAGTGAAATTTAAAAAG 
               
               
                   
               
               
                 hCT2270768-Ex24 
                 TCCCAGTTTGTATGCTATTGAGAG 
                 CTTTGGGCCTTTTTCATTCC 
                 GCGAGGCAAAACACAAAGC 
               
               
                   
               
               
                 hCT2270768-Ex25 
                 TGGAAATTCAAAAGTGTGTGG 
                 TGTCTGGCTTATTTCACACG 
                 TTGGAAATGGCTGTACCTCAG 
               
               
                   
               
               
                 hCT2270768-Ex26 
                 CACTAATGAACCCCTCAAGACTG 
                 AACTTTTGACAGCCTACTATGTGC 
                 TACTTGAGCAGCCCACAGG 
               
               
                   
               
               
                 hCT2270768-Ex27-1 
                 TCCTTGGCAAAGTGACAATC 
                 GACCATTCATGAAAGAAACAAGC 
                 AAAGGAATGAAAGTGGTTTTTGTC 
               
               
                   
               
               
                 hCT13660-Ex16 
                 CTCTCACATACAACACCATCTCC 
                 CCATGTACCGGTAACAAAAGAAG 
                 TGCAATGTAATAGTTTTCCAAGG 
               
               
                   
               
               
                 hCT13660-Ex17 
                 ATGTATCTCATTGAAAACCCAAC 
                 TGAGCTTTCTAGGATCGTACCTG 
                 CAGCAAATGAACTAAGCCACAG 
               
               
                   
               
               
                 hCT13660-Ex18 
                 TCCCAAAGTGCTGGGATTAC 
                 GCAGGAAGGTCCAACTTGTC 
                 TGCTATACTATTTGCCCACAAAAC 
               
               
                   
               
               
                 hCT13660-Ex19 
                 CCTATGACATAAATGCCAGTACAAAC 
                 ATCTTCAACTGCGAACATGC 
                 GAATGCATTTATTCAGAGATGAGG 
               
               
                   
               
               
                 hCT13660-Ex20 
                 TCTTTTGTTCAGTCAGCATCTCTC 
                 AAGCATCAATGACTACTTTAATCAAC 
                 TGCTAGACACTTGCTGGTCAC 
               
               
                   
               
               
                 hCT13660-Ex21 
                 TTGAGAATTCAGATGAGAAACCAG 
                 TCCCAAAGTGCTGGGATTAC 
                 TTGATATTAAAGTTGCACAAACTGC 
               
               
                   
               
               
                 hCT13660-Ex22 
                 GAAGGCCACTCTCAAACCTG 
                 TTGTTGCCTTTGTCATTTTG 
                 TCAATTGTGTGACATATCACCTACC 
               
               
                   
               
               
                 hCT13660-Ex23 
                 TCAAGGCTTGCATTTCATTG 
                 ATGTGACTGTGGGCAGGAAC 
                 TCACTGTAGAAATCCAAGTACCAC 
               
               
                   
               
               
                 hCT13660-Ex24 
                 TTCCACACTCCAAAGAATGC 
                 GCTGGTGAGATGTCAAAACG 
                 TCTGCATCAGTTTGATTCTGC 
               
               
                   
               
               
                 hCT13660-Ex25-1 
                 AATTGCAATCCTCTTGGTAGC 
                 TCAACATATTACTTCCTCCAGAAGTC 
                 AATGCACTTTTTATTTTATTAG 
               
               
                   
               
               
                 hCT32594-Ex66-2 
                 GCCAAGACCAAGCAACTCC 
                 TTCTCCCATGTCAGGGAATC 
                 GAAAAGTGCCGGTTCTTGAG 
               
               
                   
               
               
                 hCT32594-Ex67-1 
                 ATAAACGACCGCTGGCCTAC 
                 GACCCTCAAAGGCTAACGTG 
                 GCCTACACAGTCCGTTTTCC 
               
               
                   
               
               
                 hCT32594-Ex67-2 
                 GTACATCCGGGGACACAATG 
                 TCCCTGGTCAGCACAGACTAC 
                 AGAGGAGCGTGTGTTGCAG 
               
               
                   
               
               
                 hCT32594-Ex68 
                 ACCGGGTTCTTCCAGCTAAG 
                 AGCTGTCTCATTTCCACCATC 
                 ACTCTGACGGTGGAGCTGAG 
               
               
                   
               
               
                 hCT32594-Ex69-1 
                 CAATGCGTGCGTTAAATCTG 
                 CGCGTCGTTTATGTCAAATC 
                 GCTCTTGGTGCTAAGTTAAAGAGG 
               
               
                   
               
               
                 hCT32594-Ex69-2 
                 CCCAATGCCACGGACTAC 
                 CGCGTCGTTTATGTCAAATC 
                 ATCCAGCTGGCTCTGATAGG 
               
               
                   
               
               
                 hCT32594-Ex70 
                 ATCCAGCTGGCTCTGATAGG 
                 CATAACACACAGGGGTGCTG 
                 TGAACAGCCAGATCCTCTCC 
               
               
                   
               
               
                 hCT32594-Ex71 
                 CTGGTGCTGAAACTCGACTG 
                 GAACTGGGCGAGGTTGTG 
                 GTCCCACCTTGTTAGGAAGC 
               
               
                   
               
               
                 hCT32594-Ex72-1 
                 GTCTCGTTCTCTCCCTCACG 
                 TCCCTTTCTTACACGCAAAC 
                 TGGCATTCTGAAAACGGTTC 
               
               
                   
               
               
                 hCT32594-Ex72-2 
                 CACAACCTCGCCCAGTTC 
                 CAGTTCCGCCTGTACATTCAC 
                 GCAAACAGCCTGGACAATC 
               
               
                   
               
               
                 hCT7976-Ex5 
                 AGCATCACCCTCAGAGCATAC 
                 AGCGCTCCTGCTTTCAGTC 
                 CACATATTTCTGTCCCCTGTTG 
               
               
                   
               
               
                 hCT7976-Ex6 
                 TGCCATACCTCTTAGGCACTTC 
                 GTCTTGGCGCAGATCATCAC 
                 TGTGGTTCTTTGGAGCACAG 
               
               
                   
               
               
                 hCT7976-Ex7 
                 CGACAGAGCAAGATTCCATC 
                 TTTTGTCACCAGTTGAAATGC 
                 CCAAGGTACATTTCGGAAAAC 
               
               
                   
               
               
                 hCT7976-Ex8 
                 AGATTGCCATCTGAGGAAGG 
                 GACTGGGAAAAAGCATGAGC 
                 ACCAGCCCTTTCCTCTTGTC 
               
               
                   
               
               
                 hC17976-Ex9 
                 GCATGGAGAGGAAGTGAACC 
                 CGGTGATCATAATATTGTCATTGTG 
                 TTCTTCCTCATGCCATTGTG 
               
               
                   
               
               
                 hCT7976-Ex10 
                 TGGCCAGAGAGTTTGATTTATG 
                 GGAAGTGTGGGCTTGTCTTC 
                 GTGGCATCTGGCTGTCATC 
               
               
                   
               
               
                 hCT7976-Ex11-1 
                 CCCTCAATCTCTTGGGAAAG 
                 TGCACAGTCCATCCTTTGTC 
                 CAATTAGTTTTCCTTGAGCACTCC 
               
               
                   
               
               
                 hCT7976-Ex11-2 
                 TGGTTTCTTCTCATGGACAGG 
                 AATGCCAGCTTTCACAATGTC 
                 TCTTCTTTATCCAGGACATCTGTG 
               
               
                   
               
               
                 hCT7448-Ex21 
                 GGGTGTCCACACTTCTCAGG 
                 GGCCAAGACCACATGGTAAG 
                 CCTGGGAGAGGTCTGGTTC 
               
               
                   
               
               
                 hCT7448-Ex22 
                 CCGGAAGAAACAATGAGCAG 
                 TCCTACATTAAGACAGCATGGAAC 
                 GGCAGCATCTTGGTCTGAAG 
               
               
                   
               
               
                 hCT7448-Ex23 
                 GGTGTGAGCTGAGTGAGCAG 
                 TGCCTCCCTTTTAAGGCTATC 
                 GAGCACTTGGGAGACCTGAG 
               
               
                   
               
               
                 hCT7448-Ex24 
                 GTGGGAATGACCTTCCTTTC 
                 AGGTCCTTCTGCCAACAAAG 
                 AGGGAAGCATGAGCACAGTC 
               
               
                   
               
               
                 hC17448-Ex25 
                 GGATGAACAGGCAGATGTGAG 
                 CGTCTTCTCTCCTCCAATGC 
                 TGAGTTCTGTCTGGCTGTGG 
               
               
                   
               
               
                 hCT7448-Ex26 
                 AGCCCCTTCTATCCAGTGTG 
                 GGTATTCAGTTGGGGCTCAG 
                 TGATGAGGGATGAGGGAAAC 
               
               
                   
               
               
                 hCT7448-Ex27 
                 TGCCCACAGCATCTGTCTAC 
                 TGTATCCACGTGGTCAGCTC 
                 AGGGTTAGGGAGCCTAGCTG 
               
               
                   
               
               
                 hCT7448-Ex28-1 
                 ATTGTGTGCCAGTCATTTGC 
                 ACAGGACGCTCGGTCAAC 
                 TCCTTGGAACACCCCTGTC 
               
               
                   
               
               
                 hCT1951523-Ex39-2 
                 TTCCACATTAAGCATGAGCAC 
                 TTGCCATCAGTACAAATGAGTTTAG 
                 CAGTCATGATACCTACACTTCCATC 
               
               
                   
               
               
                 hCT1951523-Ex40 
                 GACAGTCATTCTTTTCATAGGTCATAG 
                 TTCCTGCTTTTTAAGAGTGATCTG 
                 CAACTCTGAAATAAAAGCAATCTGG 
               
               
                   
               
               
                 hCT1951523-Ex41 
                 CCACATAGTAAGCCTTCAATGAC 
                 AGGAAGGAAGGGATGGAAAC 
                 TTCTTTGGTTATGAAATGAACAATC 
               
               
                   
               
               
                 hCT1951523-Ex42 
                 TGAAAAATGTTCCTTTATTCTTG 
                 AGAAACCACTCATGAAAA 
                 TTGAATAAAAGTAGATGTTTCTTGTCC 
               
               
                   
               
               
                 hCT1951523-Ex43 
                 TCTGAGAACATTCCCTGATCC 
                 CGCATTACTACATGATCCACTG 
                 TACCAAGAATATAATACGTTGTTATGG 
               
               
                   
               
               
                 hCT2257127-Ex76 
                 TCAGCTCTCTAATCCTGAACTGC 
                 TGTCACAGAAAGCATGAGACC 
                 CGGCTTCTGGCACATAAAAC 
               
               
                   
               
               
                 hCT2257127-Ex77-1 
                 AGCAGAGAAGAAACATATACCAT 
                 AGAAATAACTGTCAATATCCCAGTATCAC 
                 CCATTGAGCACTCCATTCATTAC 
               
               
                   
               
               
                 hCT2257127-Ex77-2 
                 CATTTTGGGAAAGGAGGTTC 
                 TCATTAAACATTTAGTAATGTGTGCTC 
                 CCCTGGGAATCTGAAAGAATG 
               
               
                   
               
               
                 hCT2257127-Ex78 
                 ATTACAGGCGTGAGCCACTG 
                 AGGCAACAGGGCAAGACTC 
                 TGGGCCGTTGTCTCATATAC 
               
               
                   
               
               
                 hCT2257127-Ex79-1 
                 TTTGGCACTGTCTTCAGAGG 
                 CCTGAAAGGGAGAATAAAAGG 
                 CACTCTGGCTTTCCCTCTG 
               
               
                   
               
               
                 hCT2257127-Ex79-2 
                 AGAGGGAACACCCTTTCCTG 
                 CCTGAAAGGGAGAATAAAAGG 
                 AGGTCATGAATGGGATCCTG 
               
               
                   
               
               
                 hCT2257127-Ex80 
                 TATAGCGTTGTGCCCATGAC 
                 TATTGACCCAGCCAGCAGAC 
                 CATATTGCTTGGCGTCCAC 
               
               
                   
               
               
                 hCT2257127-Ex81 
                 TCCTGCCTCTTTGCTATTTTTCAATG 
                 TATATTGAGACTCAAATATCGA 
                 TCTTGGTGATCTTTGCCTTTG 
               
               
                   
               
               
                 hCT2257127-Ex82 
                 TTGCCTCAGAGAGATCATCAAG 
                 TGATGCATATCAGAGCGTGAG 
                 TCATCAAGATTATTCGATATTTGAGTC 
               
               
                   
               
               
                 hCT2257127-Ex83-1 
                 TAGGGGCGCTAATCGTACTG 
                 TTCAATGACCATGACAAAACG 
                 CGAGAAAGTAAAGTGCCTGCTG 
               
               
                   
               
               
                 hCT2257127-Ex83-2 
                 TCTGATATGCATCAGCCACTG 
                 TTCAATGACCATGACAAAACG 
                 CGGGATTGGAGACAGACATC 
               
               
                   
               
               
                 hCT2257127-Ex84 
                 TGATTTCAAGGGAAGCAGAG 
                 TGGTTTTCAAGCAGACAATCC 
                 GAGGATGCTGCCATTTGTG 
               
               
                   
               
               
                 hCT2257127-Ex85 
                 TGTAGAAAGCAAGGCTGCTC 
                 TCCTCCTCAATGAAAGCAGAG 
                 CATGCTAACAGAGTGTCAAGAGC 
               
               
                   
               
               
                 hCT1951422-Ex19 
                 ACCCCAAAGTCATCCAAGTG 
                 CAATGTGATCCCAACTGGTC 
                 CGAATTCTTTTTGCCATTTC 
               
               
                   
               
               
                 hCT1951422-Ex20 
                 AAAGGCTCCAGTTGATGGAC 
                 TTATTGCCAATTGGAGTTTGG 
                 AAAGTCTGCAAGGGGCTATG 
               
               
                   
               
               
                 hCTI951422-Ex21 
                 CCATTAAAACCACTCTAAGTCAGG 
                 TTCTGTTGGCTTATCATTTTTG 
                 TCAGGCTAGAAATGTATCCAAGG 
               
               
                   
               
               
                 hCT1951422-Ex22 
                 AAGCCTCCTCCAGAAAAGAAG 
                 CCCAGAAACTAAATAAAATGCAG 
                 AAAGGAAAGGGGTAATCCAG 
               
               
                   
               
               
                 hCT1951422-Ex23 
                 CCCTCCTGTCCACTGAGATG 
                 AATCAAATTTGTTGCATTAAAAATC 
                 TTTACTTTTTATGATTACCTCTGATGC 
               
               
                   
               
               
                 hCT1951422-Ex24 
                 TCTCAAGCTGCCTCACAATG 
                 GTTTTCTCATTCCTTTCTCTTCC 
                 AAAGAAAATTCAAATGAAAATAAGTCG 
               
               
                   
               
               
                 hCT1951422-Ex25 
                 AAAGACATTGCCATGCAAAC 
                 TTTGGGAAAGGGAACACAAG 
                 CATGCAAACTTGGGTCTAGATG 
               
               
                   
               
               
                 hCT1951422-Ex26 
                 TTGTTGGGCTCCAAATAAAC 
                 GATTTTTCCTTGGAACATCCTC 
                 TTGGCTTTTCCCCTCATAC 
               
               
                   
               
               
                 hCT13051-Ex5 
                 CCCTGGAGTGCTTACATGAG 
                 CGGGGATCAGATTTGCTATG 
                 TAAAGCCTTTCCCAGCTCAG 
               
               
                   
               
               
                 hCT13051-Ex6 
                 GACTTTATAAACACTCGACATTAGAGC 
                 TAGGGGGTCATCCTCAGGTG 
                 CCTGCTGCTTCCACAGGAC 
               
               
                   
               
               
                 hCT13051-Ex7 
                 ATGATGACCTCTGGCAGGAC 
                 GTCTTCCCCTGCTCAATCAC 
                 CATGGACGTCCTGTGGAAG 
               
               
                   
               
               
                 hCT13051-Ex8 
                 GAATCAACCGTCAGCGTGTC 
                 GACACGTTGTGGGCCAGCCAGT 
                 GTGTCCCATTCATCCTCACC 
               
               
                   
               
               
                 hCT13051-Ex9 
                 CTGGCACCGGGGAAAACAGAG 
                 CTGCCGGTTATCTTCGGACACGTT 
                 AACAGAGGAGGCGCTGAAG 
               
               
                   
               
               
                 hCT2282983-Ex40 
                 TGGACATCGACTACAAGTCTGG 
                 TGAGTGAGGGCAGACAGATG 
                 GCCTCACCCTACCCATCC 
               
               
                   
               
               
                 hCT2282983-Ex41 
                 TCCTTGGGGTTTTGAAGAAG 
                 TGGCACCTGAACCATGTAAG 
                 AGATTGCTGGGGTTCCTTTC 
               
               
                   
               
               
                 hCT2282983-Ex42 
                 AAGGCCTTCCAGACTCTTGC 
                 CGTACATGCCGAAGTCTGTC 
                 CCACCTCACTCCATCTCTGG 
               
               
                   
               
               
                 hCT2282983-Ex43 
                 CCTCTTTGTTTTTCCCTACCG 
                 GCCCTGGTTTTAACCCTTAAC 
                 TGGGGTAAGTTCCCTGAGTG 
               
               
                   
               
               
                 hCT2282983-Ex44-1 
                 CTTCCACAGTGGGGGTACAG 
                 CCAGCTCCAGCTTCTGACTC 
                 TACAGAGCCAGGGAGAGTGC 
               
               
                   
               
               
                 hCT2282983-Ex44-2 
                 GACACAACGGCAACATTATGCTG 
                 TTGTGTTTTCTTGGAGACAG 
                 TATCATCCACATCGGTCAGC 
               
               
                   
               
               
                 hCT2292935-Ex46 
                 CATTCCAAAGCATCTGGTTTTAC 
                 CAATGAGCATGGGAGAGATG 
                 TTTGGGACAAGTAATTGTTATTAGC 
               
               
                   
               
               
                 hCT2292935-Ex47 
                 TTGTGAGGAACGTGTGATTAGG 
                 TGGAGTTTCTGGGACTACAGG 
                 TTGAATGCAGTGGTGCTCTC 
               
               
                   
               
               
                 hCT2292935-Ex48 
                 CTGGGCAACAGAGCAAGAC 
                 CCTTCTTCAAAGCTGATTCTCTC 
                 TCTGCCTGTGTTCTGAGCTG 
               
               
                   
               
               
                 hCT2292935-Ex49 
                 TCCCTTCTCCTTTGGCTATG 
                 CGCTCTACAGCCAATCACAG 
                 GAACTCAGCTCTGCCTGGAC 
               
               
                   
               
               
                 hCT2292935-Ex50 
                 ATAGCACCACTGCCTTCCAG 
                 TGGCATCACAATCAATAGGG 
                 GCGAGACTCGGTCTCAAAAG 
               
               
                   
               
               
                 hCT2292935-Ex51 
                 TGCAGAAGTGGAGGTGGAG 
                 CTCCAAGGGGGTTAGAGTCC 
                 ATCGTTTGCCAACTCCTAGC 
               
               
                   
               
               
                 hCT2292935-Ex52 
                 AACCCAAGCTGCTTCCTTTC 
                 CAGGAAACCAGGTCAGAAGTG 
                 AATCAGTGCAGGTGATGCAG 
               
               
                   
               
               
                 hCT2292935-Ex53 
                 AGTCCTGCCCTGATTCCTTC 
                 TTTTTGCAGAAAGGGGTCTTAC 
                 ACATGGCCTGTGTCTGCTTC 
               
               
                   
               
               
                 hCT2292935-Ex54 
                 CCCACCCACTTATTCCTGAG 
                 GCCCACCCCACTCTAGAAAC 
                 GACTGGAAGAAAATAACCAAGTTTC 
               
               
                   
               
               
                 hCT2292935-Ex55 
                 TTTCCCCTTTAGGGTAGGTAGG 
                 TGGAACCTTTTCTGCTCAAAG 
                 GGCAGGCGTTAAAGGAATAG 
               
               
                   
               
               
                 hCT2292935-Ex56 
                 CGGACATAGAGGAAGGATTGC 
                 AGCTGCATGGTGCCAAAG 
                 AAAAACAGGGCACCCATTG 
               
               
                   
               
               
                 hCT2292935-Ex57 
                 TGGCCAAACTTTTCAAATCC 
                 ATAACAATGGGCACATGCAG 
                 TTAAGCCCACAGGGAACAAG 
               
               
                   
               
               
                 hCT2292935-Ex56-1 
                 TGGGAGAGCTCAGGGAATAC 
                 GGTCATTCTTCCATCAGCAAG 
                 TGTGAGACCTTGGCCTTTTC 
               
               
                   
               
               
                 hCT2273636-Ex35-1 
                 TCCCAAAGTGCTGGGATTAC 
                 CACACCCACACTCAGACAAAG 
                 TCTTCTGAAAAATGGAGGAAGTC 
               
               
                   
               
               
                 hCT2273636-Ex35-2 
                 TTGGCTGCCATGACTAACAC 
                 GGCACTGCAGGCTAATAATG 
                 GCTCTTCCTGGGGAAGTCTC 
               
               
                   
               
               
                 hCT2273636-Ex36-1 
                 GCTCTCAGTGTGCCTCATGG 
                 GGGACGTCAAGTCTTTTCCTTC 
                 CAGTTTTTGACTGCCACTGC 
               
               
                   
               
               
                 hCT2273636-Ex36-2 
                 AAGAAACACCCCGGTTCC 
                 GGGACCTCAAGTCTTTTCCTTG 
                 TCCATGCTCGACACTATTCTG 
               
               
                   
               
               
                 hCT2273636-Ex37-1 
                 AAATTTAGTTGAGTAATGAGAGAATGC 
                 GGAAGGGAAGGAGGACAAAC 
                 TTCTACTTTACATACAAAAGGCACTC 
               
               
                   
               
               
                 hCT2273636-Ex37-2 
                 GTAAAATTGGCCCTGCTTTG 
                 CGTCTCAAACTACCAAGTCTGG 
                 AGTTGGGCTTAGCCTGGATG 
               
               
                   
               
               
                 hCT2273636-Ex38 
                 CATAACCACATGCAGCAACC 
                 CACCCAGTGCTGTTTCAATG 
                 AGTATCACGTCCATGTTGGAG 
               
               
                   
               
               
                 hCT2273636-Ex39 
                 AATTGGCCTTGGAGACAGAC 
                 CGCCGCATAATGTGTAAAAC 
                 CAATGTTTGCTTTGAAAAAGG 
               
               
                   
               
               
                 hCT2273636-Ex40-1 
                 TTCATGTGAGCAGGTATGCTG 
                 TGCCATATTTAACTGCCATTTC 
                 TGAGCAAAACCTGTGGAATG 
               
               
                   
               
               
                 hCT2273636-Ex40-2 
                 TTGTGTACGACCCTCTGGTG 
                 TGCCATATTTAACTGCCATTTC 
                 TTTGCTGGTGCTGTCTATGG 
               
               
                   
               
               
                 hCT2273636-Ex41 
                 TTTGTACAGTGGAGGCAACG 
                 GCAGTCACTGAGACAGCTTTTATC 
                 GGATGTGCAAAATGTTCTTCTG 
               
               
                   
               
               
                 hCT7084-Ex17 
                 CAGCTGGTTATGTGTGTTTATGG 
                 TAAGCATAGCCTCGGAGAAC 
                 GGGAGCAGGTGTTATTGATTG 
               
               
                   
               
               
                 hCT7084-Ex18 
                 TGTCCTCATGGTTGCTTTTC 
                 GGACCATTAATAGCTACCTTCCTG 
                 GGTGAGGAGTTTTCCCAAGC 
               
               
                   
               
               
                 hCT7084-Ex19 
                 CAGGGACATGCTATCCAAAG 
                 AGGCAAGACAACATATTTGAAAG 
                 AGCAGAGAGTTTGTTAATGTTTTTAG 
               
               
                   
               
               
                 hCT7084-Ex20 
                 TGGTGGAACTTGTGTTTTTCC 
                 AAGGGCTATGTGTCATTTTGTTC 
                 GCTGACTTCTATTGGGAGCATAC 
               
               
                   
               
               
                 hCT7084-Ex21 
                 TCATACGGTTTTGGCAGCTC 
                 CATCAAGCAAGCAAACAAATG 
                 CAGAGGTATGGTTTGGGTCTC 
               
               
                   
               
               
                 hCT7084-Ex22 
                 ACAGAGGGAGAAGGGCTCAG 
                 AATTCCCCCAAAAGCTTCC 
                 TGGGGGTCTAGGACTATGGAG 
               
               
                   
               
               
                 hCT7084-Ex23 
                 TGGGACAATTTCGCAGAAG 
                 TTCCCTCCTGGCTAAGAACC 
                 GCTGTGTTTTCTTAATTTCCTGTATG 
               
               
                   
               
               
                 hCT7084-Ex24-1 
                 ATGAAAGCATGCTGCCTGATG 
                 AAAAGCAGAGGGAATCATCG 
                 CAGCCTCCTGCAGACTTTG 
               
               
                   
               
               
                 hCT2257641-Ex1-56 
                 GGGGGCCTTTAGAAGGAAG 
                 TCCCATTCATGACCTGGAAG 
                 CATTTTGGGAAAGGAGGTTC 
               
               
                   
               
               
                 hCT2257641-Ex1-57 
                 TGGAGTTCCTGAGAAATGAGC 
                 GGCCCGCTTTAAGAGATCAG 
                 CGGTCAGTATGACGGTAGGG 
               
               
                   
               
               
                 hCT2257641-Ex1-58 
                 AGAGGGAACACCCTTTCCTG 
                 CATGCCCAAAGTCGATCC 
                 AGGTCATGAATGGGATCCTG 
               
               
                   
               
               
                 hCT2257641-Ex1-59 
                 CATGATGTTGGAGCTTACATGC 
                 ACACATCCATGGTGTTGGTG 
                 GGCGCTAATCGTACTGAAAC 
               
               
                   
               
               
                 hCT2257641-Ex1-60 
                 CGGGATTGGAGACAGACATC 
                 TGCCACAGCCACATAGTCTC 
                 TATGGTGGCCATGGAGACTG 
               
               
                   
               
               
                 hCT2257641-Ex1-61 
                 CATCATGGTACACGCACTCC 
                 TTCTATCTGCAGACTCCCACAG 
                 AGGAGCCCTCCTTTGATTG 
               
               
                   
               
               
                 hCT29277-Ex55 
                 CTCAATCAGAGCCTGAACCAC 
                 GGAAAAGAAAGCAGGAGAAGC 
                 GGCCAGTGGTATCTGCTGAC 
               
               
                   
               
               
                 hCT29277-Ex56 
                 CCCGGCCTAAAGTTGTAGTTC 
                 AAATGGAGAAAAGCCTGGTTC 
                 AAGACAAAATCCCAAATAAAGCAG 
               
               
                   
               
               
                 hCT29277-Ex57 
                 TGGGAGACTGTCAAGAGGTG 
                 AAGCAATCCTCCCACCTTG 
                 ATTGGTTTGAGTGCCCTTTG 
               
               
                   
               
               
                 hCT29277-Ex58 
                 TTCCTCCAAGGAGCTTTGTC 
                 CCTTCCTTTTTCACTCACACAC 
                 AAAATGCTTTGCACTGACTCTG 
               
               
                   
               
               
                 hCT29277-Ex59 
                 TTCCCTGTCCAGACTGTTAGC 
                 TGATTTAATAATGAAGATGGGTTGG 
                 TTCATCTTTATTGCCCCTATATCTG 
               
               
                   
               
               
                 hCT29277-Ex60 
                 CCGGTTATGCACATCATTTAAG 
                 ACTCAGTACCCCAGGCAGAG 
                 TTAAAGATTATACCAAGTCAGTGGTC 
               
               
                   
               
               
                 hCT29277-Ex61 
                 GCAGCCAGAGCAGAAGTAAAC 
                 TCAAACTCCTGGGCTCAAAC 
                 CATGTGGTTTCTTGCCTTTG 
               
               
                   
               
               
                 hCT29277-Ex62 
                 TCTAATGAAAGCCCACTCTGC 
                 CAGCCACATCCCCCTATG 
                 AAGCATAGGCTCAGCATACTACAC 
               
               
                   
               
               
                 hCT29277-Ex63 
                 AAGTGTGCATGATGTTTGTTCC 
                 TGCCTTCTTCCACTCCTTTC 
                 CCCATCAACTACCATGTGACTG 
               
               
                   
               
               
                 hCT29277-Ex64-1 
                 GATGACCAAGAATGCAAACG 
                 AAGAGTGAAAGCAGAGATGTTCC 
                 GGTCCTGTTGTCAGTTTTTCAG 
               
               
                   
               
               
                 NM_005026 Ex17 
                 ATCATCTTTAAGAACGGGGATGG 
                 ACTAAGCCTCAGGAGCAGCCT 
                 GGTCCTGGGGTGCTCCTAGA 
               
               
                   
               
               
                 NM_005026 Ex18 
                 CCTCAGATGCTGGTGCCG 
                 GATACTTGGGGAAGAGAGACCTACC 
                 TCCTCAACTGAGCCAAGTAGCC 
               
               
                   
               
               
                 NM_005026 Ex19 
                 TCTTCATGCCTTGGCTCTGG 
                 GAGGGGAGAGGAGGGGGAG 
                 TGTGTCCTCCATGTTCTGTTGG 
               
               
                   
               
               
                 NM_005026 Ex20 
                 TCCGAGAGAGTGGGCAGGTA 
                 CACAAACCTGCCCACATTGC 
                 TGGCCCCTCTGCCTAGCA 
               
               
                   
               
               
                 NM_005026 Ex21 
                 GGGCAGGTTTGTGGGTCAT 
                 CCTGGGCGGCTCAACTCT 
                 CCACTGCTGGGTCCTGGG 
               
               
                   
               
               
                 NM_005026 Ex22 
                 GGAACTGGGGGCTCTGGG 
                 AGGCGTTTCCGTTTATGGC 
                 GAATAGAGAGCTTTTCCTGAGATGC 
               
               
                   
               
               
                 hCT1640694-Ex1-1 
                 GTTTCTGCTTTGGGACAACCAT 
                 CTGCTTCTTGAGTAACACTTACG 
                 GATTCATCTTGAAGAAGTTGATGG 
               
               
                   
               
               
                 hCT1640694-Ex1-2 
                 CTCCACGACCATCATCAGG 
                 GATTACGAAGGTATTGGTTTAGACAG 
                 ACTTGATGCCCCCAAGAATC 
               
               
                   
               
               
                 hCT1640694-Ex1-3 
                 CCCCCTCCATCAACTTCTTC 
                 GGTGTTAAAAATAGTTCCATAGTTCG 
                 CTCAAGAAGCAGAAAGGGAAG 
               
               
                   
               
               
                 hCT1640694-Ex2-1 
                 TCATCAAAAATTTGTTTTAACCTAGC 
                 TATAAGCAGTCCCTGCCTTC 
                 TCTACAGAGTTCCCTGTTTGC 
               
               
                   
               
               
                 hCT1640694-Ex2-2 
                 TTCTGAACGTTTGTAAAGAAGCTG 
                 TATAAGCAGTCCCTGCCTTC 
                 GCTGTGGATCTTAGGGACCTC 
               
               
                   
               
               
                 hCT1640694-Ex3-1 
                 GCAGCCCGCTCAGATATAAAC 
                 CTGGGCGAGAGTGAGATTCC 
                 AAAAAGCATTTCTGATATGGATAAAG 
               
               
                   
               
               
                 hCT1640694-Ex3-2 
                 TCTGAAAATCAACCATGACTGTG 
                 ATGAACCCAGGAGGCAGAG 
                 TCGAAGTATGTTGCTATCCTCTG 
               
               
                   
               
               
                 hCT1640694-Ex4-1 
                 TCTTGTGCTTCAACGTAAATCC 
                 CGGAGATTTGGATGTTCTCC 
                 AAAATAATAAGCATCAGCATTTGAC 
               
               
                   
               
               
                 hCT1640694-Ex4-2 
                 TCTCAACTGCCAATGGACTG 
                 CGGAGATTTGGATGTTCTCC 
                 TTATTCCAGACGCATTTCCAC 
               
               
                   
               
               
                 hCT1640694-Ex5 
                 TAGTGGATGAAGGCAGCAAC 
                 TTTGTAGAAATGGGGTGTTGC 
                 TTTGAGTCTATCGAGTGTGTGC 
               
               
                   
               
               
                 hCT1640694-Ex6 
                 TGCCTTTTCCAATCAATCTC 
                 AATTCCTGAAGCTCTCCCAAG 
                 TTCCTGTTTTTCGTTTGGTTG 
               
               
                   
               
               
                 hCT1640694-Ex7 
                 GGGGAAAAAAGGAAAGAATGG 
                 TGCTGAACCAGTCAAACTCC 
                 TGAATTTTCCTTTTGGGGAAG 
               
               
                   
               
               
                 hCT1640694-Ex8 
                 TTTGCTGAACCCTATTGGTG 
                 TTGCAATATTGGTCCTAGAGTTC 
                 TGGATCAATCCAAATAAAGTAAGG 
               
               
                   
               
               
                 hCT1640694-Ex9 
                 GATTGGTTCTTTCCTGTCTCTG 
                 CCACAAATATCAATTTACAACCATTG 
                 TTGCTTTTTCTGTAAATCATCTGTG 
               
               
                   
               
               
                 hCT1640694-Ex10 
                 ACCTTTTGAACAGCATGCAA 
                 TGGAAATAATGTTAAGGGTGTTTTT 
                 TATTTCATTTATTTATGTGGAC 
               
               
                   
               
               
                 hCT1640694-Ex11 
                 AAAACACCCTTAACATTATTTCCATAG 
                 TCTGCATGGCCGATCTAAAG 
                 GAAGTTAAGGCAGTGTTTTAGATGG 
               
               
                   
               
               
                 hCT1640694-Ex12 
                 TTATTCTAGATCCATACAACTTCCTTT 
                 AAAGTTGAGAAGCTCATCACTGGTAC 
                 ACCAGTAATATCCACTTTCTTTCTG 
               
               
                   
               
               
                 hCT1640694-Ex13 
                 CTGAAACTCATGGTGGTTTTG 
                 TGGTTCCAAATCCTAATCTGC 
                 TTTATTGGATTTCAAAAATGAGTG 
               
               
                   
               
               
                 hCT1640694-Ex14 
                 GAGTGTTGCTGCTCTGTGTTG 
                 TTGAGGGTAGGAGAATGAGAGAG 
                 TCTCATGTGAGAAAGAGATTAGCAG 
               
               
                   
               
               
                 hCT1640694-Ex15 
                 GGATTCCTAAATAAAAATTGAGGTG 
                 CATGCATATTTCAAAGGTCAAG 
                 TGGCTTTCAGTAGTTTCATGG 
               
               
                   
               
               
                 hCT1640694-Ex16 
                 TTGCTTTCCTGAAGTTTCTTTTG 
                 TCAAGTAAGAGGAGGATATGTCAAAG 
                 CATGTGATGGCGTGATCC 
               
               
                   
               
               
                 hCT1640694-Ex17 
                 GGGGAAAGGCAGTAAAGGTC 
                 CATCAAATATTTCAAAGGTTGAGC 
                 AGGAATACACAAACACCGACAG 
               
               
                   
               
               
                 hCT1640694-Ex18 
                 TCCTTATTCGTTGTCAGTGATTG 
                 GTCAAAACAAATGGCACACG 
                 TGCACCCTGTTTTCTTTTCTC 
               
               
                   
               
               
                 hCT1640694-Ex19 
                 CATGGTGAAAGACGATGGAC 
                 TTACAGGCATGAACCACCAC 
                 TGGACAAGTAATGGTTTTCTCTG 
               
               
                   
               
               
                 hCT1640694-Ex20-1 
                 TGGGGTAAAGGGAATCAAAAG 
                 CCTATGCAATCGGTCTTTGC 
                 TGACATTTGAGCAAAGACCTG 
               
               
                   
               
               
                 hCT1640694-Ex20-2 
                 TTGCATACATTCGAAAGACC 
                 GGGGATTTTTGTTTTGTTTTG 
                 TTGTTTTGTTTTGTTTTT 
               
               
                   
               
               
                   1 SEQ ID NO: 6 to 165 (forward primers) 
               
               
                   2 SEQ ID NO: 166 to 325 (reverse primers) 
               
               
                   3 SEQ lED NO: 326 to 485 (sequencing primers) 
               
             
          
         
       
     
       Example 2 
     This Example Demonstrates the Striking Clustering of Mutations within the PIK3CA Gene 
       [0036]    All coding exons of PIK3CA were then analyzed in an additional 199 colorectal cancers, revealing mutations in a total of 74 tumors (32%) (Table 3 and examples in  FIG. 1 ). 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 PIK3CA mutations in human cancers 
               
             
          
           
               
                   
                 Tumor type #   
               
             
          
           
               
                   
                 PIK3CA mutations* 
                 Functional 
                 Medullo- 
                   
               
             
          
           
               
                 Exon 
                 Nucleotide 
                 Amino acid 
                 domain 
                 Colon 
                 GBM 
                 Gastric 
                 Breast 
                 Lung 
                 Pancreas 
                 blastomas 
                 Adenomas 
                 Total 
               
               
                   
               
             
          
           
               
                 Exon 1 
                 C112T 
                 R38C 
                 p85 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 1 
                 G113A 
                 R38H 
                 p85 
                 2 
                   
                   
                   
                   
                   
                   
                   
                 2 
               
               
                 Exon 1 
                 G263A 
                 R88Q 
                 p85 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 1 
                 C311G 
                 P104R 
                 p85 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 1 
                 G317T 
                 G106V 
                 p85 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 1 
                 G323C 
                 R108P 
                 p85 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 1 
                 del332-334 
                 delK111 
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 2 
                 G353A 
                 G118D 
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 2 
                 G365A 
                 G122D 
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 2 
                 C370A 
                 P124T 
                   
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 4 
                 T1035A 
                 N345K 
                 C2 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 4 
                 G1048C 
                 D350H 
                 C2 
                   
                 1 
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 5 
                 T1132C 
                 C378R 
                 C2 
                   
                 1 
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 7 
                 T1258C 
                 C420R 
                 C2 
                 2 
                   
                   
                   
                   
                   
                   
                   
                 2 
               
               
                 Exon 7 
                 G1357C 
                 E453Q 
                 C2 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 9 
                 C1616G 
                 P539R 
                 Helical 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 9 
                 G1624A 
                 E542K 
                 Helical 
                 9 
                   
                   
                   
                   
                   
                   
                 1 
                 10 
               
               
                 Exon 9 
                 A1625G 
                 E542G 
                 Helical 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 9 
                 A1625T 
                 E542V 
                 Helical 
                   
                   
                   
                   
                   
                   
                   
                 1 
                 1 
               
               
                 Exon 9 
                 G1633A 
                 E545K 
                 Helical 
                 21 
                   
                   
                   
                 1 
                   
                   
                   
                 22 
               
               
                 Exon 9 
                 A1634G 
                 E545G 
                 Helical 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 9 
                 G1635T 
                 E545D 
                 Helical 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 9 
                 C1636A 
                 Q546K 
                 Helical 
                 5 
                   
                   
                   
                   
                   
                   
                   
                 5 
               
               
                 Exon 9 
                 A1637C 
                 Q546P 
                 Helical 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 12 
                 C1981A 
                 Q661K 
                 Helical 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 13 
                 A2102C 
                 H701P 
                 Helical 
                   
                 1 
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 18 
                 G2702T 
                 C901F 
                 Kinase 
                 1 
                   
                 1 
                   
                   
                   
                   
                   
                 2 
               
               
                 Exon 18 
                 T2725C 
                 F909L 
                 Kinase 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 20 
                 T3022C 
                 S1008P 
                 Kinase 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 20 
                 A3073G 
                 T1025A 
                 Kinase 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 20 
                 C3074A 
                 T1025N 
                 Kinase 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Exon 20 
                 G3129T 
                 M1043I 
                 Kinase 
                 2 
                   
                   
                   
                   
                   
                   
                   
                 2 
               
               
                 Exon 20 
                 C3139T 
                 H1047Y 
                 Kinase 
                 2 
                   
                   
                   
                   
                   
                   
                   
                 2 
               
               
                 Exon 20 
                 A3140G 
                 H1047R 
                 Kinase 
                 15 
                   
                 2 
                 1 
                   
                   
                   
                   
                 18 
               
               
                 Exon 20 
                 A3140T 
                 H1047L 
                 Kinase 
                 1 
                   
                   
                   
                   
                   
                   
                   
                 1 
               
               
                 Bran 20 
                 G3145A 
                 G1049S 
                 Kinase 
                   
                 1 
                   
                   
                   
                   
                   
                   
                 1 
               
             
          
           
               
                 Tumors with mutations 
                 74 
                 4 
                 3 
                 1 
                 1 
                 0 
                 0 
                 2 
                   
               
               
                 No. samples screened 
                 234 
                 15 
                 12 
                 12 
                 24 
                 11 
                 12 
                 76 
               
               
                 Percent of tumors with mutations 
                 32% 
                 27% 
                 25% 
                 8% 
                 4% 
                 0% 
                 0% 
                 3% 
               
               
                   
               
               
                 *Exon number with nucleotide and amino acid change resulting from mutation. Nucleotide position refers to position within coding sequence, where position 1 corresponds to the first position of the start codon. Functional domains are described in FIG. 1 legend. 
               
               
                   # Number of non-synonymous mutations observed in indicated tumors. Colon, colorectal cancers; GBM, glioblastomas; gastric, gastric cancers; breast, breast cancers; lung, lung cancers; pancreas, pancreatic cancers; medulloblastomas; adenomas, benign colorectal tumors. All mutations listed were shown to be somatic except for five colorectal cancers and one glioblastoma where no corresponding normal tissue was available. Mutations were identified in 58 of 201 mismatch repair (MMR) proficient colorectal cancers, and 16 of 33 MMR-deficient colorectal cancers. Some tumors with PIK3CA mutations contained mutations in KRAS or BRAF while others did not, suggesting that these genes operate through independent pathways. Seven tumors contained two somatic alterations. In addition to the 92 nonsynonymous mutations recorded in the table, we detected 3 synonymous alterations. 
               
             
          
         
       
     
       Example 3 
     This Example Demonstrates that the Mutations in PIK3CA Occur Late in Tumorigenesis 
       [0037]    To determine the timing of PIK3CA mutations during neoplastic progression, we evaluated 76 pre-malignant colorectal tumors of various size and degree of dysplasia. 
         [0038]    Only two PIK3CA mutations were found (E542K and E542V), both in very advanced adenomas greater than 5 cm in diameter and of tubuluvillous type. These data suggest that PIK3CA abnormalities occur at relatively late stages of neoplasia, near the time that tumors begin to invade and metastasize. 
       Example 4 
     This Example Demonstrates that PIK3CA Mutations in a Variety of Different Cancer Types 
       [0039]    We then evaluated PIK3CA for genetic alterations in other tumor types (Table 1). Mutations were identified in four of fifteen (27%) glioblastomas, three of twelve (25%) gastric cancers, one of thirteen (8%) breast, and one of twenty four (4%) lung cancers. No mutations were observed in eleven pancreatic cancers or twelve medulloblastomas. In total, 89 mutations were observed, all but 3 of which were heterozygous. 
       Example 5 
     This Example Demonstrates the Non-Random Nature of the Genetic Alterations Observed 
       [0040]    The sheer number of mutations observed in PIK3CA in five different cancer types strongly suggests that these mutations are functionally important. This conclusion is buttressed by two additional independent lines of evidence. First, analysis of the ratio of non-synonymous to synonymous mutations is a good measure of selection during tumor progression, as silent alterations are unlikely to exert a growth advantage. The ratio of non-synonymous to synonymous mutations in PIK3CA was 89 to 2, far higher than the 2:1 ratio expected by chance (P&lt;1×10 −4 ). Second, the prevalence of non-synonymous changes located in the PI3K catalytic and accessory domains was 120 per Mb tumor DNA, over 100 times higher than the background mutation frequency of nonfunctional alterations observed in the genome of cancer cells (P&lt;1×10 −4 ) (9). 
         [0041]    Although the effect of these mutations on kinase function has not yet been experimentally tested, their positions and nature within PIK3CA imply that they are likely to be activating. No truncating mutations were observed and &gt;75% of alterations occurred in two small clusters in exons 9 and 20 (Table 2 and  FIG. 1 ). The affected residues within these clusters are highly conserved evolutionarily, retaining identity in mouse, rat, and chicken. The clustering of somatic missense mutations in specific domains is similar to that observed for activating mutations in other oncogenes, such as RAS (10), BRAF (11, 12), β-catenin (13), and members of the tyrosine kinome (14). 
         [0042]    These genetic data suggest that mutant PIK3CA is likely to function as an oncogene in human cancers. 
       Example 6 
     This Example Demonstrates that Gene Amplification of PIK3CA is not Common 
       [0043]    Quantitative PCR analysis of PIK3CA in 96 colorectal cancers showed no evidence of gene amplification, suggesting that gene copy alterations are not a significant mechanism of activation in this tumor type. The primers used were:
       Real time PI3K hCT1640694 20-1F (intron)       
 
         [0000]                            TTACTTATAGGTTTCAGGAGATGTGTT;   (SEQ ID NO: 486)                
and
       Real time PI3K hCT1640694 20-1R         
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                 GGGTCTTTCGAATGTATGCAATG 
                 (SEQ ID NO: 487) 
                   
               
             
          
         
       
     
         [0046]    The Sequence Listing appended to the end of this application contains the following sequences:
       SEQ ID NO: 1=coding sequence only (nt 13 to 3201 of SEQ ID NO: 2)   SEQ ID NO: 2=RNA sequence (NM — 006218)   SEQ ID NO: 3=protein sequence (NP — 006209)   SEQ ID NO: 4=exon 9   SEQ ID NO: 5=exon 20   SEQ ID NO: 6 to 165=forward primers   SEQ ID NO: 166 to 325=reverse primers   SEQ ID NO: 326 to 485=sequencing primers   SEQ ID NO: 486 and 487 amplification primers       
 
       REFERENCES AND NOTES 
       [0000]    
       
         1. R. Katso et al.,  Annu Rev Cell Dev Biol  17, 615-75 (2001). 
         2. I. Vivanco, C. L. Sawyers,  Nat Rev Cancer  2, 489-501 (July, 2002). 
         3. W. A. Phillips, F. St Clair, A. D. Munday, R. J. Thomas, C. A. Mitchell,  Cancer  83, 41-7 (Jul. 1, 1998). 
         4. E. S. Gershtein, V. A. Shatskaya, V. D. Ermilova, N. E. Kushlinsky, M. A. Krasil&#39;nikov,  Clin Chim Acta  287, 59-67 (September, 1999). 
         5. B. Vanhaesebroeck, M. D. Waterfield,  Exp Cell Res  253, 239-54 (Nov. 25, 1999). 
         6. S. Djordjevic, P. C. Driscoll,  Trends Biochem Sci  27, 426-32 (August, 2002). 
         7. Catalytic subunits of PI3Ks were identified by analysis of InterPro (IPR) PI3K domains (IPR000403) present within the Celera draft human genome sequence. This resulted in identification of 15 PI3Ks and related PI3K genes. The kinase domain of PIK3CD gene was not represented in the current draft of human genome sequence and was therefore not included in this study. 
         8. Sequences for all annotated exons and adjacent intronic sequences containing the kinase domain of identified PI3Ks were extracted from the Celera draft human genome sequence (URL address: www host server, domain name celera.com). Celera and Genbank accession numbers of all analyzed genes are available in Table 1. Primers for PCR amplification and sequencing were designed using the Primer 3 program (URL address: http file type, www-genome.wi.mit.edu host server, cgi-bin domain name, primer directory, primer3_www.cgi subdirectory), and were synthesized by MWG (High Point, N.C.) or IDT (Coralville, Iowa). PCR amplification and sequencing were performed on tumor DNA from early passage cell lines or primary tumors as previously described (12) using a 384 capillary automated sequencing apparatus (Spectrumedix, State College, Pa.). Sequence traces were assembled and analyzed to identify potential genomic alterations using the Mutation Explorer software package (SoftGenetics, State College, Pa.). Of the exons extracted, 96% were successfully analyzed. Sequences of all primers used for PCR amplification and sequencing are provided in Table S1. 
         9. T. L. Wang et al.,  Proc Natl Acad Sci USA  99, 3076-80. (2002). 
         10. J. L. Bos et al.,  Nature  327, 293-7 (1987). 
         11. H. Davies et al.,  Nature  (Jun. 9, 2002). 
         12. H. Rajagopalan et al.,  Nature  418, 934. (2002). 
         13. P. J. Morin et al.,  Science  275, 1787-90 (1997). 
         14. A. Bardelli et al.,  Science  300, 949 (May 9, 2003). 
         15. J. Li et al.,  Science  275, 1943-7 (1997). 
         16. P. A. Steck et al.,  Nat Genet.  15, 356-62 (1997). 
         17. T. Maehama, J. E. Dixon,  J Biol Chem  273, 13375-8 (May 29, 1998). 
         18. M. P. Myers et al.,  Proc Natl Acad Sci USA  95, 13513-8 (Nov. 10, 1998). 
         19. L. Shayesteh et al.,  Nat Genet.  21, 99-102 (January, 1999). 
         20. J. Q. Cheng et al.,  Proc Natl Acad Sci USA  89, 9267-71 (Oct. 1, 1992). 
         21. L. Hu, J. Hofmann, Y. Lu, G. B. Mills, R. B. Jaffe,  Cancer Res  62, 1087-92 (Feb. 15, 2002). 
         22. J. Luo, B. D. Manning, L. C. Cantley, Cancer Cell 4, 257-62 (2003).

Technology Category: 8