WO2006052249A1 - Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate - Google Patents

Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate Download PDF

Info

Publication number
WO2006052249A1
WO2006052249A1 PCT/US2004/037288 US2004037288W WO2006052249A1 WO 2006052249 A1 WO2006052249 A1 WO 2006052249A1 US 2004037288 W US2004037288 W US 2004037288W WO 2006052249 A1 WO2006052249 A1 WO 2006052249A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
polypeptide
tumor
phosphorylated
akt
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2004/037288
Other languages
English (en)
Inventor
Charles L. Sawyers
Paul S. Mischel
Bradley L. Smith
Katherine Crosby
George V. Thomas
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cell Signaling Technology Inc
University of California Berkeley
University of California San Diego UCSD
Original Assignee
Cell Signaling Technology Inc
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cell Signaling Technology Inc, University of California Berkeley, University of California San Diego UCSD filed Critical Cell Signaling Technology Inc
Priority to PCT/US2004/037288 priority Critical patent/WO2006052249A1/fr
Priority to US11/667,430 priority patent/US20080299589A1/en
Publication of WO2006052249A1 publication Critical patent/WO2006052249A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57557—Immunoassay; Biospecific binding assay; Materials therefor for cancer of other specific parts of the body, e.g. brain
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites

Definitions

  • the present invention provides methods for the examination of biochemical pathways that are shown to be disregulated in pathologies such as cancer and to reagents adapted for performing these methods.
  • deregulation of the PI3K/Akt and Ras/MAPK pathways occurs in many types of cancer (see, e.g., Vivanco et al., Nat Rev Cancer. 2: 489-501., 2002), including glioblastoma (GBM) and cancer of the prostate (see, e.g., Vivanco et al., Nat Rev Cancer. 2: 489-501, 2002; Feldkamp et al., Journal of Neurooncology 35: 223-248, 1997; Mischel et al., Brain Pathology, Jan;13(l):52-61 2003).
  • GBM glioblastoma
  • GBM Glioblastoma multiforme
  • the tumor suppressor gene PTEN is altered in 30-40% of GBMs (see, e.g., Liu et al., Cancer Res. 57: 5254-7., 1997; Schmidt et al., J Neuropathol Exp Neurol. 58: 1170-83., 1999; Smith et al., J Natl Cancer Inst. 93: 1246-56., 2001).
  • PTEN protein deficient GBMs would show coordinated activation of this pathway.
  • Primary GBMs (those that arise as de novo grade IV tumors) also commonly over-express the oncogene EGFR, and its variant EGFRvIII, which activate signaling through both the RAS/MAPK and PI3K/Akt pathways. Therefore, it is also possible that EGFR and EGFRvIII expressing GBMs would show coordinate activation of the ERK and the Akt pathways. To date however, the relationship between these various pathways has not been delineated.
  • PTEN tumor suppressor gene Inactivation of the PTEN tumor suppressor gene is also implicated in the development of both primary and metastatic prostate cancers.
  • Conditional deletions of PTEN in the mouse prostate leads to cancer and many human prostate cancer cell lines and xenografts have PTEN deletions or point mutations (see, e.g. Wang et al., Cancer CeU, 4: 209-221, 2003; Trotman et al., PLoS Biol, 1: E59, 2003; Steck et al., Nat Genet, 15: 356-362, 1997; Pesche et al., Oncogene, 16: 2879-2883, 1998; and Teng et al., Cancer Res, 57: 5221-5225, 1997).
  • PTEN phosphatidyl inositol 3-kinase
  • PI3K phosphatidyl inositol 3-kinase
  • PI3K is a lipid kinase that phosphorylates phosphatidylinositols at the 3-position (Ptdlns [3, 4, 5] P3), which subsequently recruit kinases such as AKT (a potent oncogenic survival factor), leading to a cascade of constitutive activation of downstream effectors (Figure 4), including the mammalian Target Of Rapamycin (mTOR) (Vivanco et al.,.
  • mTOR mammalian Target Of Rapamycin
  • Deregulated activation of the PBK/ Akt pathway is common in a variety of different cancers including glioblastomas and cancers of the prostate, bile duct, bladder, breast, colon, endometrium, blood, liver, lung, skin, ovary, pancreas and thyroid to name a few. Consequently, the assessment of this pathway can be used to obtain diagnostic, prognostic and therapeutic information, for example to stratify patients for targeted kinase inhibitor therapy.
  • the disclosure provided herein identifies a series of biomarkers that are associated with deregulated activation of the PI3K/Akt pathway as well as optimized methods for examining these markers. Significantly, the disclosed methods for examining these markers are useful with a wide variety of tissue samples including formalin fixed, paraffin embedded biopsy samples.
  • a series of PBK/ Akt pathway biomarkers associated with cancers such as prostate cancer and glioblastoma multiforme can be examined using for example a series of antibodies such as phospho-specific antibodies.
  • a mammalian cell such as a cancer cell derived from a biopsy sample can be examined for evidence of PI3K/Akt pathway activation by examining a tissue sample containing this cell for the presence of: a phosphorylated S6 polypeptide (SEQ ID NO: 1); a phosphorylated mTOR polypeptide (SEQ ID NO: 2); a phosphorylated FKHR polypeptide (SEQ ID NO: 3); a phosphorylated AKT polypeptide (SEQ ID NO: 4); a phosphorylated ERK polypeptide (SEQ ID NO: 8); or decreased levels of expression of the PTEN polypeptide (SEQ ID NO: 5), wherein the presence of a phosphorylated S6, mTOR, FKHR,
  • the cell is examined for the presence of a plurality of characteristics such as a phosphorylated S6 polypeptide (SEQ ID NO: 1) and decreased levels of expression of the PTEN polypeptide (SEQ ID NO: 5).
  • Certain embodiments of the invention comprise further methodological steps, the step of using the results of the examination to identify and/ or assess a therapeutic agent that may be used to treat the cancer such as the step of using the results of the examination to evaluate the effect of an mTOR inhibitor such as rapatnycin or an analogue thereof or an EGFR inhibitor such as ZD-1839 or an analogue thereof on a cancer cell.
  • One preferred embodiment of the invention is a method for identifying a mammalian glioma tumor likely to respond or responsive to an EGFR polypeptide (SEQ ID NO: T) inhibitor or an mTOR polypeptide (SEQ ID NO: 2) inhibitor, the method comprising examining a sample obtained from the tumor for: the expression of PTEN polypeptide (SEQ ID NO: 5); and the presence of at least one of, a phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1); a EGFR polypeptide (SEQ ID NO: 7); a phosphorylated AKT polypeptide (SEQ ID NO: 4); and a phosphorylated ERK polypeptide (SEQ ID NO: 8), wherein decreased expression of PTEN polypeptide together with decreased phosphorylation of S6 ribosomal polypeptide in the sample, as compared to a control, identifies the glioma tumor as likely to respond or responsive to an mTOR inhibitor, and wherein decreased
  • Another preferred embodiment of the invention is a method for identifying a mammalian prostate tumor likely to respond or responsive to an EGFR polypeptide (SEQ ID NO: 7) inhibitor or an mTOR polypeptide (SEQ ID NO: 2) inhibitor, the method comprising examining a sample obtained from the tumor for: the expression of PTEN polypeptide (SEQ ID NO: 5); and the presence of at least one of, a phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1); a EGFR polypeptide (SEQ ID NO: 7); a phosphorylated AKT polypeptide (SEQ ID NO: 4); and a phosphorylated ERK polypeptide (SEQ ID NO: 8), wherein decreased expression of PTEN polypeptide together with decreased phosphorylation of S6 ribosomal polypeptide in the sample, as compared to a control, identifies the prostate tumor as likely to respond or responsive to an mTOR inhibitor, and wherein decreased expression an of PTEN together with normal
  • kits for characterizing a mammalian tumor or cell comprising: an antibody that binds PTEN (SEQ ID NO: 5); and/ or an antibody that binds phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1); and/or an antibody that binds EFGR (SEQ ID NO: 7); and/or an antibody that binds phosphorylated AKT (SEQ ID NO: 4); and/or an antibody that binds phosphorylated ERK (SEQ ID NO: 8).
  • the kit further includes an antibody that binds Ki-67 polypeptide (SEQ ID NO: 9), and/or p-H3 histone polypeptide (SEQ ID NO: 10) and/or caspase-3 polypeptide (SEQ ID NO: 11).
  • the kit further comprises a secondary antibody which binds to one of the primary antibodies directed to these polypeptides.
  • the kit comprises a plurality of antibodies that bind to the various polypeptides.
  • Figure 1 shows the itnmunohistochemical expression of PTEN, p-Akt, p- mTOR, p-FKHR and p-S6 in GBM tumor samples.
  • A Representative images demonstrating PTEN protein loss in tumors cells with retention of staining in vascular endothelium (0), diminished PTEN staining relative to the endothelium (1), and no evidence of PTEN protein loss (2). NC is the negative control.
  • Figure 2 shows the immunohistochemical expression of EGFR, EGFRvIII and p-Erk in GBM tumor samples.
  • A Representative images demonstrating diffuse EGFR, EGFRvIII and p-Erk positivity (+). Representative images of tumors lacking EGFR, EGFRvIII and p-ERK expression ate also shown (-). NC represents the negative controls.
  • Figures 3A and 3B provide an illustration of the interaction between members of the PI3K/Akt pathway and kinase inhibitors in GBM tumor samples.
  • Figure 3A shows that rapamycin inhibits S6 phosphorylation in glioblastoma in vivo.
  • Figure 3B shows that the rapamycin-mediated inhibition of S6 phosphorylation correlates with diminished tumor proliferation.
  • Ki-67 a marker of cellular proliferation was used to assess whether lapamycin-mediated inhibition of S6 had an effect on tumor growth.
  • Figure 4 provides a schematic demonstrating the components of the
  • Figures 5A-5E provide a validation of PTEN and the phospho-specific antibodies.
  • LY 2940002 resulted in decreased staining intensity of p-AKT, p-FKHR and p-S6K.
  • the staining intensity of p- ERK before and after treatment remained unchanged.
  • p-ERK staining was examined in HT129 cells, by the addition of EGF (lOOng/ml for 20 minutes). This staining is subsequently reduced when a MEK inhibitor, UO-126 (20 ⁇ M for 20 minutes) is added to the induced cells, in contrast to lack of inhibition by LY 294002.
  • E LNCaP cells were grown in culture and exposed to either vehicle or the mTOR inhibitor, CCI- 779 (2OnM) for 18 hours and subsequently prepared as described in the material and methods section.
  • Sections were stained with p-AKT and p-S6.
  • the addition of CCI-779 resulted in decreased staining intensity of p-S6 but had no effect on p-AKT, which is consistent with mTOR being downstream of AKT but upstream of p-S6.
  • Figures 6A and 6B provide a definition of PTEN scoring criteria.
  • Figure 7 provides a demonstration of the inter-relationships between signaling molecules in vivo. MDS analysis shows that PTEN protein and AKT phosphorylation are tightly linked, confirming what is known of their in vitro relationship.
  • mammal for purposes of treatment or therapy refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.
  • cancer refers to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • examples of cancer include but are not limited to astrocytoma, blastoma, carcinoma, glioblastoma, leukemia, lymphoma and sarcoma.
  • cancers include breast cancer, ovarian cancer, colon cancer, colorectal cancer, rectal cancer, squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, Hodgkin's and non-Hodgkin's lymphoma, testicular cancer, esophageal cancer, gastrointestinal cancer, renal cancer, pancreatic cancer, glioblastoma, cervical cancer, glioma, liver cancer, bladder cancer, hepatoma, endometrial carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
  • Growth inhibition when used herein refers to the growth inhibition of a cell in vitro and/ or in vivo. The inhibition of cell growth can be measured by a wide variety of methods known in the art.
  • Classical M-phase blockers include the vincas (vincristine and vinblastine), TAXOL®, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
  • Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
  • Such agents further include inhibitors of cellular pathways associated with disregulated cell growth such as the PI3K/Akt pathway. Further information can be found in The Molecular Basis of Cancer. Mendels
  • Treatment refers to both therapeutic treatment and prophylactic or preventative measures.
  • the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit ⁇ i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/ or relieve to some extent one or more of the symptoms associated with the disorder.
  • the drug may prevent growth and/ or kill existing cancer cells, it may be cytostatic and/ or cytotoxic.
  • efficacy in vivo can, for example, be measured by assessing tumor burden or volume, the time to disease progression (TTP) and/ or determining the response rates (RR).
  • antibody is used in the broadest sense and specifically covers single monoclonal antibodies and antibody compositions with polyepitopic specificity (e.g. polyclonal antibodies) as well as antibody fragments so long as retain their ability to immunospecifically recognize a target polypeptide epitope.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, Le., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiting production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature.
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al.. J. MoL Biol, 222:581-597 (1991), for example.
  • polynucleotide means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/ or RNA. In the art, this term if often used interchangeably with “oligonucleotide”.
  • a polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T) can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, » in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).
  • polypeptide means a polymer of at least about 10 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "protein”.
  • inhibitor encompasses molecules capable of inhibiting one or more of the biological activities of target molecules such as mTOR and/ or EGFR polypeptide.
  • Illustrative inhibitors include the targeted small-molecule inhibitors and antibody inhibitors disclosed herein as well as other inhibitors known in the art such as anti-sense polynucleotides and siRNA. Consequently one skilled in the art will appreciate that such inhibitors encompass molecules which inhibit both polynucleotide synthesis and/ or function (e.g. antisense polynucleotide molecules) as well those which inhibit polypeptide synthesis and/ or function (e.g. molecules which block phosphorylation and hence activity of a target polypeptide such as mTOR).
  • Deregulated activation of the PI3K/Akt pathway is common in a variety of different cancers (see, e.g. Fresno Vara et al., Cancer Treat Rev.. 30(2): 193-204 (2004; Mitsiades et al., Curr. Cancer Drug targets, 4(3): 235-256 (2004); Brader et al., Tumori, 90(1): 2-8 (2004); and Sansal et al., J. Clin. Oncol., 22(14): 2954-2963 (2004).
  • cancers that exhibit deregulated activation of the PI3K/Akt pathway include glioblastomas and cancers of the prostate (see, e.g., Vivanco et al., Nat Rev Cancer. 2: 489-501, 2002; Feldkamp et al., Journal of Neurooncology 35: 223-248, 1997; Mischel et al., Brain Pathology, Jan;13(l):52-61 2003) as well as cancers of the bile duct (see, e.g. Tanno et al., Cancer Res., 64(10): 3486-3490 (2004)), bladder (see, e.g.
  • the disclosure provided herein identifies a series of biomarkers that are associated with deregulated activation of the PI3K/Akt pathway as well as optimized methods for examining these markers. Consequently, the disclosure provided herein allows the examination of this pathway in cancer. Significantly, the disclosed methods for examining these markers are useful with a wide variety of tissue samples including formalin fixed, paraffin embedded biopsy samples. Various aspects of this' disclosure are described in Choe et al., Cancer Res. 2003 Jun l;63(ll):2742-6. [0032] As noted above, the disclosure provided herein identifies a series of biotnarkers that are associated with deregulated activation of the PI3K/Akt pathway, a pathway whose deregulated activation is common in cancers such as gliomas.
  • the disclosure provided herein further provides optimized methods for examining these biomarkers. Consequently, the disclosure allows the examination of the activation status of these biomarkers in a variety of cancers. As the art that teaches that this growth related pathway is common pathway that is disregulated in a wide variety of human cancers, artisans understand that the methods and materials disclosed herein can be universally applied to examine this pathway in all cancers in which the deregulated activation of the PBK/ Akt pathway is observed.
  • the disclosed methods for examining these biomarkers are useful with a wide variety of tissue samples including formalin fixed, paraffin embedded biopsy samples.
  • these markers can be examined using a panel of antibodies such as phospho-specific antibodies.
  • a mammalian cell such as a cell derived from a formalin fixed, paraffin embedded glioblastoma multiforme biopsy sample can be examined for evidence of Akt pathway activation by examining a tissue sample containing this cell for the presence of the various target molecules disclosed herein including phosphorylated polypeptides.
  • Certain embodiments of the invention identify and/ or assess a therapeutic agent that may be used to treat the glioblastoma such as rapamycin or an analogue thereof or an EGFR inhibitor such as ZD-1839 or an analogue thereof.
  • the invention disclosed herein provides methods and immunohistochemical reagents that can be used to identify the activation state of the PI3K/Akt signaling pathway in clinical samples such as prostate and glioblastoma biopsy • • samples. These methods and reagents identify a coordinate regulation of the Akt/mTOR signaling pathway in response to loss of the PTEN tumor suppressor gene. As specific kinase inhibitors that target this pathway are currently in development (see, e.g., Neshat et al., Proc Natl Acad Sci U S A. 98: 10314-9, 2001), and further because this mutation is common in glioblastoma and prostate cancer, this disclosure provides an important clinical tool for selecting patients for appropriate therapy.
  • the invention can be practiced by performing immunohistochemical analysis on routinely processed patient biopsy samples.
  • the results of these assays can be used as criteria for inclusion in clinical trials, and to assess outcome differences in patients in which this pathway is deregulated.
  • the methods and reagents disclosed herein can be used to determine the activation state of biomarker polypeptides such as Akt and its downstream effectors such as mTOR, ERK, Forkhead and 86-kinase on routinely processed patient biopsy samples (e.g. glioblastoma samples) and this information can be used to select patients for therapy with targeted pathway inhibitors.
  • PIP3 catalyzes the activation of the Akt kinase (and its downstream effectors mTOR, forkhead and S6-kinase), which promote cell proliferation and survival.
  • the PTEN tumor suppressor gene encodes a phosphatase that removes the phosphate group from PIP3, thereby regulating the activation state of this pathway. PTEN loss results in constitutive signaling through PIP3, and hence unregulated activation of the Akt pathway.
  • PTEN is lost in many types of cancer including glioblastomas and cancers of the prostate.
  • the Akt pathway is dysregulated in many other cancers.
  • PTEN-defLcient cancer cells are dramatically more sensitive to inhibition of the Akt pathway at the level of mTOR (see, e.g., Neshat et al., Proc Natl Acad Sci U S A. 98: 10314-9, 2001), than PTEN wild-type cells, including non-cancerous cells. Therefore, mTOR inhibitors can be a highly selective and effective therapy for patients whose tumors have PTEN loss and Akt pathway activation.
  • PI3'K/Akt pathway activation can be detected in routinely processed patient biopsies.
  • PTEN loss is significantly correlated with Akt activation, which is significantly associated with activation of downstream effectors mTOR, S6 and FKHR.
  • PTEN loss is not the only mechanism of PI3'K/Akt pathway activation, and demonstrated that EGFR and EGFRvIII co-expression are significantly associated with activation of this pathway.
  • PI3K/Akt and Erk pathway activation have significant impact on cancer progression and survival.
  • the epidermal growth receptor factor receptor contributes to the malignant phenotype of human glioblastomas (see, e.g. Thomas et al., Int J Cancer. 2003 Mar 10;l 04(1): 19-27).
  • Studies in SKMG-3 cells a GBM cell line that maintains EGFR gene amplification in vitro demonstrate that EGF treatment stimulated phosphorylation of the EGFR as well as the downstream effectors Erk, AKTl, stat3 and c-Cbl. Under minimal growth conditions, unstimulated SKMG-3 cells contain constitutively phosphorylated Erk and AKTI.
  • the EGFR kinase inhibitor PD158780 reduces the constitutive phosphorylation of the receptor and Erk but not that of AKTl .
  • inhibition of phosphatidylinositol-3-kinase (PI3K) blocks the constitutive phosphorylation of Erk and AKT-I but not the EGFR.
  • PI3K phosphatidylinositol-3-kinase
  • EGFR appears to play an important role in the pathogenesis of colorectal cancer as shown for example by studies of the EGFR tyrosine kinase inhibitor ZDl 839 in metastatic colorectal cancer patients in which serial biopsies were taken pre- and posttreatment to assess biological activity (see, e.g. Daneshmand CUn Cancer Res. 2003 Jul;9(7):2457-64). In these studies, paired biopsies were obtained from colorectal cancer patients before and after treatment. Posttreatment samples showed a statistically significant reduction in cancer cell proliferation.
  • PI3'K/Akt pathway is commonly deregulated in GBMs, but its identification in routine biopsies has presented a challenge. In the face of new kinase inhibitors that target this pathway, the need for an assay that can be used to stratify patients for therapy has become critical. As disclosed herein, we demonstrate that activation of the PI3'K/Akt pathway can be detected by immunohistochemistry using a panel of phospho-sp 1 ecific antibodies. We show that 38% of untreated primary GBMs demonstrate evidence of PTEN protein loss, and that this is significantly associated with Akt activation. We further demonstrate that phosphorylation of Akt is significantly correlated with phosphorylation of downstream effectors mTOR, FKHR and S6.
  • PI3'K/Akt pathway activation can be detected in paraffin-embedded biopsy samples, and provides evidence that PTEN loss is highly correlated with Akt pathway activation in primary GBMs. These results also provide evidence that co-expression of EGFR and EGFRvIII can activate the PI3'K pathway in GBMs with normal PTEN immunohistochemical exptession. The results further provide evidence that activation of these signaling pathways has considerable impact on GBM patient progression and survival.
  • the disclosure provided herein specifically demonstrates that the activation of the PI3'K/Akt pathway can be detected with phospho-specific antibodies in routinely processed patient biopsies.
  • the disclosed methods and materials are used to examine glioblastomas.
  • the disclosed methods and materials are used to examine cancers of the prostate.
  • PTEN-deficient GBMs have coordinated activation of the Akt pathway and its downstream effectors mTOR, FKHR and S6.
  • GBMs co-expressing EGFR and EGFRvIII have activation of the PI3'K/Akt and Erk signaling pathways.
  • tissue microarray from 45 untreated primary GBM patient biopsies and analyzed the immunohistochemical expression of p-Akt and downstream effectors p- mTOR, p-FKHR and p-S6, as well as p-Erk.
  • EGFR, EGFRvIII expression, and PTEN loss all of which can promote activation of the PI3'K/Akt pathway, were also analyzed and association with PI3'K/Akt and Erk pathway activation were determined. The prognostic implications of PI3'K/Akt and Erk pathway activation were also analyzed.
  • Immunohistochemical analysis requires a subjective determination by pathologists. Proteomic approaches have the potential to be a more objective and sensitive methods and may become clinically feasible in the future (see, e.g., Iiotta et al., Jama. 286: 2211-4., 2001; Liotta et al., Breast Cancer Res. 2: 13-4, 2000; Petricoin et al., Lancet. 359: 572-7., 2002; Petricoin et al., Nat Rev Drug Discov. 1: 683- 95., 2002).
  • Akt Akt can be detected by immunohistochemistty done on patient biopsies, and it has been suggested that it may have biological or prognostic implications (see, e.g., Gupta et al., Clin Cancer Res. 8: 885-92., 2002; Malik et al., Clin Cancer Res. 8: 1168-71., 2002).
  • convergent inputs to mTOR, FKHR and S6 downstream of Akt, or in and Akt-independent fashion may play an important role in modulating the biological behavior of GBMs.
  • concurrent Erk and Akt-mediated signaling may be required for optimal activation of p70 S6 kinase, and formation of p-S6 (see, e.g., Iijima et al., J Biol Chem. 277: 23065-75., 2002; Shi et al., J Biol Chem. 277: 15712-20., 2002).
  • GBMs are among the most heterogeneous tumors, as has been previously shown (see, e.g., Cheng et al., J Neuropathol Exp Neurol. 58: 120-8., 1999; Jung et al., J Neuropathol Exp Neurol. 58: 993-9., 1999).
  • Using the disclosure provided herein and methods typically employed in the art one can directly determine the extent of intra-tumor molecular heterogeneity for PTEN, EGFR and EGFRvIII and assess the impact of this on pathway activation, prognosis and response to therapy.
  • gliomas cellular pathways in the family of tumors termed "gliomas”. Briefly, the brain contains two major cell types: neurons and glia. Glial cells give rise to the family of tumors termed “gliomas”. There are several distinct types of tumors within this glioma grouping. These can range from very benign, slow-growing tumors to rapidly enlarging, highly malignant cancerous types. The most commonly occurring tumors within the glioma family are astocytomas, oligodendroglioma and ependymomas. In addition, some patients may have tumors with a mixed appearance. Astrocytomas are the most common type of glioma.
  • astrocytomas can be located either superficially or deep within the brain and can affect critical structures. As they arise from the astrocyte cells (which serve as supporting elements of the brain), astrocytomas are generally infiltrative in nature.
  • grade I tumors are the least malignant. These tumors grow slowly and microscopically appear almost normal; surgery alone may be effective. Grade I tumors are often associated with long-term survival. Grade II tumors grow slightly faster than grade I tumors and have a slightly abnormal microscopic appearance. These tumors may invade surrounding normal tissue, and may recur as a grade II or higher tumor. Grade III tumors are malignant. These tumors contain actively reproducing abnormal cells and invade surrounding normal tissue. Grade III tumors frequently recur, often as grade IV tumors. Grade IV tumors are the most malignant and invade wide areas of surrounding normal tissue.
  • Low-grade astrocytomas are termed benign and occur generally in children or young adults. These tumors carry a better prognosis than higher grade astrocytomas. Although the management of these low-grade astrocytomas can be controversial, those tumors which are surgically accessible are usually resected.
  • One of the concerns with low-grade astrocytomas in adults is that they can undergo a malignant transformation and change into a higher-grade, or malignant tumor. The methods of the invention can be used to monitor such transformations.
  • astrocytomas grade I normal karyotype is observed most frequently; among the cases with abnormal karyotypes, the most frequent chromosomal abnormalities loss of the X and Y sex- chromosomes; loss of 22q is found in 20-30% of astrocytomas; other abnormalities observed in low grade tumors include gains on chromosome 8q, 1Op, and 12p, and losses on chromosomes Ip, 4q, 9p, lip 16p, 18 and 19.
  • Anaplastic astrocytomas are more aggressive tumors and, as such, are usually treated in a more radical fashion.
  • chromosome gains or losses are frequent: trisomy 7 (the most frequent), loss of chromosome 10, loss of chromosome 22, loss of 9p, 13q; other abnormalities, less frequently described are: gains of chromosomes Iq, Hq, 19, 20, and Xq.
  • Glioblastoma multiforme is the most malignant form of astrocytomas. Although these tumors can occur at almost any age, the peak incidence is between 50 and 70 years old. Glioblastoma multiforme (GBM) is also called a high- grade glioma and is graded by pathologists as Grade TV /TV astrocytoma. These tumors mostly occur in adults with the peak incidence between 50 and 70 years of age. Generally the time from the onset of symptoms to diagnosis is relatively short, usually just a few weeks.
  • Glioblastomas typically show several chromosomal changes: by frequency order, gain of chromosome 7 (50-80% of glioblastomas), double minute chromosomes, total or partial monosomy for chromosome 10 (70% of tumors) associated with the later step in the progression of glioblastomas partial deletion of 9p is frequent (64% of tumors): 9pter-23; partial loss of 22q in 22ql3 is frequently reported loss or deletion of chromosome 13, 13ql4-q31 is found in some glioblastomas trisomy 19 was reported in glioblastomas by cytogenetic and comparative genomic hybridization (CGH) analysis; the loss of 19q in 19ql3.2-qter was detected by loss of heterozygosity (LOH) studies in glioblastomas deletion of chromosome 4q, complete or partial gains of chromosome 20 has been described; gain or amplification of 12ql4-q21 has been reported the loss of chromosome Y might
  • Oligodendrogliomas are benign, slow growing tumors that occur usually in young adults. Often these are located within the frontal lobes which can allow for a safe, complete operative resection. Many oligodendrogliomas contain calcium (little specks of bone) seen best on CT scans. [0057] In addition to glioblastomas, the methods of the invention are applicable to a wide variety of other cancers where disregulation of the PI3K/Akt pathway is associated with a concurrent disregulation in cellular growth. For example, in yet another illustrative embodiment of the invention, the disclosed methods and materials are used to examine cancers of the prostate.
  • phospho-specific antibodies disclosed herein were also used to follow changes in phosphorylation status of these proteins in a prostate cancer cell line, before and after treatment with CCI-779. This highlights the utility of these antibodies to confirm target inhibition in patient samples post treatment.
  • typical embodiments of the invention examine cellular pathways in the family of tumors termed "prostate cancers".
  • cancers of the prostate are typically categorized into stages. In stage Tl (Stage A), the tumor is located within the prostate only. It causes no symptoms and is too small to be felt during a digital rectal exam (DRE) or to be seen on an imaging scan. These tumors are usually found by chance during surgery for some other prostate disease, or by following up on screening tests that measure PSA.
  • DRE digital rectal exam
  • stage T2 the tumor is still located within the prostate but is large enough to be felt during a DRE. There are often no symptoms.
  • Stage T3 the tumor has spread from the prostate into the immediately surrounding tissue, possibly including the seminal vesicles.
  • stage T4 the tumor is still within the pelvic region but may have spread farther to areas of the bladder or rectum.
  • stage JV+ Stage D1
  • prostate cancer is described as N+ if prostate cancer cells are detected in the lymph nodes in the pelvic area.
  • stage M+ Stage D2
  • a tumor that is M+ has spread beyond the pelvic area to other parts of the body. Bone pain, weight loss, and tiredness are common symptoms.
  • Gleason grading system The most common system used in the USA to grade the appearance of prostate cancer tissue is called the Gleason grading system. This grading system is based on a number range from 2 to 10. The lower the number, the lower the grade, and the slower the cancer is growing. The higher the score, the higher the grade of the tumor. High-grade tumors grow more quickly than low-grade tumors, and are more likely to spread to other parts of the body. Grades under 4 mean that the cancer cells look similar to your normal cells, and the cancer is likely to be less aggressive. Grades 5 to 7 are in the intermediate range.
  • Certain embodiments of the invention include methods to obtain information used to identify a therapeutic agent for treating a cancer such as a glioblastoma or a prostate cancer in a human. For example, methods of the invention examine the levels of certain polypeptides (e.g. PTEN) and/or the phosphorylation state of certain polypeptides (e.g. S 6) to obtain information on how the cancer cell will respond to rapamycin or a rapamycin analog.
  • certain polypeptides e.g. PTEN
  • S 6 the phosphorylation state of certain polypeptides
  • Rapamycin also known as sirolimus or rapammune
  • sirolimus or rapammune is a macrolide, related to cyclosporin with immunosuppressive properties and antiproliferative activity in various human tumor cells lines and tumor xenograft models.
  • rapamycin is a downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway that mediates cell survival and proliferation, and consequently is a target for anticancer therapeutic development.
  • PI3K phosphatidylinositol 3-kinase
  • Akt protein kinase B
  • rapamycin-like compounds block the actions of these downstream signaling elements, which results in cell cycle arrest in the Gl phase. Rapamycin and its analogues also prevent cyclin-dependent kinase (CDK) activation, inhibit retinoblastoma protein phosphorylation, and accelerate the turnover of cyclin Dl 3 leading to a deficiency of active CDK4/cyclin Dl complexes, all of which potentially contribute to the prominent inhibitory effects of rapamycin at the Gl /S boundary of the cell cycle.
  • CDK cyclin-dependent kinase
  • Rapamycin and rapamycin analogues have demonstrated impressive growth-inhibitory effects against a broad range of human cancers.
  • mammalian target of rapamycin mTOR
  • the methods of the invention can be used to examine the PI3K/Akt pathway and then select an appropriate therapeutic agent in cells having a deregulated PI3K/Akt pathway (e.g. rapamycin).
  • rapamycin a deregulated PI3K/Akt pathway
  • Rapamycin and its analogs see, e.g. Mita et al., Clin Breast Cancer 2003 Jun;4(2):126-37; Hosoi et al., MoI Pharmacol.
  • EGFR epidermal growth factor receptor
  • Compounds that target the extracellular ligand-binding region of EGFR include antibodies such as Cetuximab (also known as Erbitux or IMC-C225).
  • Other compounds such as tyrosine kinase inhibitors which target the intracellular domain of EGFR, include ZD-1839 (also known as gefitinib or Iressa), OSI-774 (also known as Erlotinibor or Tarceva), PD-153053, PD- 168393 and CI-1033, have been studied in clinical settings alone or in combination with radiation or chemotherapy.
  • compounds such as h-R3, ABX-EGF, EMD- 55900 and ICR-62 have proved to be effective in targeting malignant cells alone or in combination with traditional therapies.
  • ZD 1839 (Iressa) is currendy being studied in clinical trails for patients with glioblastoma multiforme.
  • the methods of the invention can be used to examine the PI3K/Akt pathway and then select an appropriate therapeutic agent in cells that do not have a deregulated PI3K/Akt pathway (e.g. an EGFR inhibitor).
  • an EGFR inhibitor e.g. an EGFR inhibitor.
  • EGFR inhibitors see, e.g. Khalil et al., Expert Rev Anticancer Ther. 2003 Jun;3(3):367-80; Chakravarti et al., Int J Radiat Oncol Biol Phys. 2003 Oct 1;57(2 Suppl):S329; Wissner et al., Bioorg Med Chem Lett.
  • invention disclosed herein has a number of embodiments.
  • Illustrative embodiments of the invention include methods which examine tumor samples such as formalin fixed, paraffin embedded glioblastoma multiforme or prostate cancer biopsy samples for evidence of deregulated activation of the PI3K/Akt pathway. These methods involve examining the presence and/ or phosphorylation status of the disclosed biomarkers that are associated with this pathway in order to identify and/or assess a therapeutic agent that may be useful in the treatment of such a cancer. As disclosed herein, the presence and/or phosphorylation status of the disclosed biomarkers serves as a marker or proxy of pathway activity.
  • the methods of the invention are used in evaluating the whether a tumor such as a prostate cancer or glioma is likely to respond (i.e. is likely to exhibit growth inhibition) when contacted with an mTOR inhibitor or an EGFR inhibitor.
  • a biomarker polypeptide that is associated with the activation of a pathway e.g. a phosphorylated S6 ribosomal polypeptide (SEQ ID NO: I)
  • SEQ ID NO: I phosphorylated S6 ribosomal polypeptide
  • the tumor is examined prior to its exposure to the inhibitor.
  • the methods evaluate whether a tumor such as a glioma or prostate cancer is responsive (i.e. exhibits growth inhibition) to an mTOR inhibitor or an EGFR inhibitor.
  • a biomarker polypeptide that is associated with the activation of a pathway e.g. a phosphorylated S6 ribosomal polypeptide (SEQ ID NO: I)
  • SEQ ID NO: I phosphorylated S6 ribosomal polypeptide
  • One such embodiment of the invention is a method for identifying a mammalian glioma (e.g. glioblastoma multiforme) tumor likely to respond, is responsive to an EGFR polypeptide (SEQ ID NO: 7) inhibitor or an mTOR polypeptide (SEQ ID NO: 2) inhibitor, the method comprising examining a sample obtained from the tumor for: the expression of PTEN polypeptide (SEQ ID NO: 5); and the presence of at least one of, a phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1); a EGFR polypeptide (SEQ ID NO: 7); a phosphorylated AKT polypeptide (SEQ ID NO: 4); and a phosphorylated ERK polypeptide (SEQ ID NO: 8), wherein decreased expression of PTEN polypeptide together with decreased phosphorylation of S6 ribosomal polypeptide in the sample, as compared to a control, identifies the glioma tumor
  • the phosphorylation of S6 ribosomal polypeptide is determined subsequent to contacting the tumor or sample with an mTOR inhibitor and/ or the phosphorylation of AKT and/ or ERK is determined subsequent to contacting the tumor or sample with an EGFR inhibitor.
  • the mTOR inhibitor is rapamycin, SDZ-RAD, CCI-779, RAD 001, or AP23573 and the EGFR inhibitor is ZD-1839, OSI-774, PD- 153053, PD-168393, IMC-C225 or CI-1033.
  • Another embodiment of the invention is a method for identifying a mammalian prostate tumor likely to respond, is responsive to an EGFR polypeptide (SEQ ID NO: 7) inhibitor or an mTOR polypeptide (SEQ ID NO: 2) inhibitor, the method comprising examining a sample obtained from the tumor for: the expression of PTEN polypeptide (SEQ ID NO: 5); and the presence of at least one of, a phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1); a EGFR polypeptide (SEQ ID NO: 7); a phosphorylated AKT polypeptide (SEQ ID NO: 4); and a phosphorylated ERK polypeptide (SEQ ID NO: 8), wherein decreased expression of PTEN polypeptide together with decreased phosphorylation of S6 ribosomal polypeptide in the sample, as compared to a control, identifies the prostate tumor as likely to respond or responsive to an mTOR inhibitor, and wherein decreased expression an of PTEN together with normal
  • ⁇ and/ or phosphorylation of ERK identifies the prostate tumor as not likely to respond and/ or is non-responsive to an EGFR inhibitor.
  • the phosphorylation of S6 ribosomal polypeptide is determined subsequent to contacting the tumor or sample with an mTOR inhibitor and/or the phosphorylation of AKT and/or ERK is determined subsequent to contacting the tumor or sample with an EGFR inhibitor.
  • the mTOR inhibitor is rapamycin, SDZ-RAD, CCI-779, RAD 001, or
  • AP23573 and the EGFR inhibitor is ZD-1839, OSI-774, PD-153053, PD-168393, IMC-
  • the expression of the biomarker polypeptides is examined using an antibody such as an antibody that binds an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 1, an antibody that binds an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 4, or an antibody that binds an epitope comprising a phosphorylated threonine residue at position 202 and tyrosine 204 in SEQ ID NO: 8.
  • the sample is a paraffin embedded biopsy sample.
  • Another embodiment of the invention is a method for identifying a mammalian tumor that does not express a PTEN polypeptide (SEQ ID NO: 5) and which is not likely to respond or is nonresponsive to an inhibitor of mTOR polypeptide (SEQ ID NO: 2) activity, the method comprising examining a sample obtained from the tumor for the presence of phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1) after contacting the tumor or the sample with the inhibitor, wherein, an observable decrease in phosphorylated S6 ribosomal polypeptide in the sample, as compared to a control that is not contacted with the inhibitor identifies the tumor as likely to respond or responsive to the inhibitor, and wherein no observable decrease in phosphorylated S6 ribosomal polypeptide in the sample, as compared to a control identifies the tumor as not likely to respond or nonresponsive to the inhibitor.
  • Yet another embodiment of the invention is a method for identifying a mammalian tumor that expresses a PTEN polypeptide (SEQ ID NO: 5) and which is not likely to respond or is nonresponsive to an inhibitor of EGFR polypeptide (SEQ ID NO: 7) activity, the method comprising examining a sample obtained from the tumor for the presence of EGFR (SEQ ID NO: 7) and the presence of a phosphorylated AKT polypeptide (SEQ ID NO: 4) or the presence of a phosphorylated ERK polypeptide (SEQ ID NO: 8) after contacting the tumor or the sample with the inhibitor, wherein an increase in the levels of the EGFR polypeptide and the levels of phosphorylated AKT polypeptide or phosphorylated ERK polypeptide identifies the tumor as not likely to respond or nonresponsive to the inhibitor.
  • the sample obtained from the tumor is examined for the presence of a phosphorylated AKT polypeptide (SEQ ID NO: 4) and the presence of a phosphorylated ERK polypeptide (SEQ ID NO: 8).
  • certain embodiments of the invention include the examination of the expression of a polypeptide or phosphorylation of a polypeptide.
  • the examination of such polypeptide expression and polypeptide phosphorylation status in a cell or tissue sample is typically evaluated as compared to a control, i.e. a control cell and/ or tissue sample that has a defined or predetermined level of polypeptide expression or phosphorylation.
  • a control can be a normal tissue (e.g. non cancerous glial cells) where it is observed that a polypeptide is typically not phosphorylated.
  • Example 3 and Figure 2 provided illustrative examples of the methods of the invention using such controls, in particular, a PTEN expression grading system known the art that uses vascular endothelium as a control. Specifically PTEN immunohistochemical staining (which is directly correlated with PTEN expression) is scored according to an established scale of 0-2, in which the vascular endothelium (score of 2) serves as an internal control.
  • Tumor cells are graded as 2 if their staining intensity is equal to that of the vascular endothelium, 1 if it is diminished relative to the vascular endothelium, and 0 if it is undetectable in the tumor cells and present in the vascular endothelium.
  • This scoring system which has been shown to be highly consistent between different cancer cell types, including gliomas (as disclosed herein) and cancers of the breast, ovary, pancreas and colon, allows artisans to readily examine the expression levels of PTEN polypeptides in a sample such as a formalin fixed, paraffin embedded biopsy sample.
  • Additional embodiments of the invention include a method for identifying a mammalian cancer cell that does not express a PTEN polypeptide (SEQ ID NO: 5) and which is likely to exhibit growth inhibition when contacted with an inhibitor of mTOR polypeptide (SEQ ID NO: 2) activity, the method comprising examining the cancer cell for the presence of phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1) after contacting the cancer cell with the inhibitor, wherein, an observable decrease in phosphorylated S6 ribosomal polypeptide in the sample, as compared to a control mammalian cancer cell that is not contacted with the inhibitor identifies the cancer cell as likely to exhibit growth inhibition when contacted with the inhibitor, and further wherein no observable decrease in phosphorylated S6 ribosomal polypeptide in the sample, as compared to a control mammalian cell identifies the cancer cell as not likely to exhibit growth inhibition when contacted with the inhibitor.
  • the inhibitor of mTOR polypeptide activity is optionally rapamycin, CCI-779, RAD 001, or AP23573.
  • the expression of the PTEN polypeptide or the presence of phosphorylated S6 ribosomal polypeptide is examined using an antibody that binds the PTEN polypeptide or the phosphorylated S6 ribosomal polypeptide (e.g. an antibody that binds an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 1).
  • the mammalian cancer cell is obtained from a paraffin embedded biopsy sample.
  • Another embodiment of the invention is a method for identifying a mammalian cancer cell that expresses a PTEN polypeptide (SEQ ID NO: 5) and which is not likely to exhibit growth inhibition when contacted with inhibitor of EGFR polypeptide (SEQ ID NO: T) activity, the method comprising examining the cancer cell for the presence of EGFR (SEQ ID NO: 7), the presence of a phosphorylated AKT polypeptide (SEQ ID NO: 4) or a the presence of a phosphorylated ERK polypeptide (SEQ ID NO: 8), wherein an increase in the levels of the EGFR polypeptide and the levels of phosphorylated AKT polypeptide or phosphorylated ERK polypeptide identifies the cancer cell as not likely to exhibit growth inhibition when contacted with inhibitor of the EGFR polypeptide.
  • the inhibitor of EGFR activity is optionally ZD-1839, OSI-774, PD-153053, PD-168393 or CI-1033.
  • the expression of the PTEN polypeptide or the presence of EGFR polypeptide is examined using an antibody that binds the PTEN polypeptide or the EGFR polypeptide.
  • the presence of phosphorylated AKT is examined using an antibody that binds an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 4 and the presence of phosphorylated ERK is examined using an antibody that binds an epitope comprising a phosphorylated threonine residue at position 202 or a phosphorylated tyrosine residue at position 204 in SEQ ID NO: 8.
  • the mammalian cancer cell is obtained from a paraffin embedded biopsy sample.
  • Another embodiment of the invention is a method for determining the responsiveness of a mammalian cancer cell to a growth inhibitory agent selected from the group consisting of a EGFR polypeptide (SEQ ID NO: 7) inhibitor or an mTOR polypeptide (SEQ ID NO: 2) inhibitor, the method comprising examining the glioblastoma cell for the presence of a S6 polypeptide (SEQ ID NO: 1) having a phosphorylated serine, threonine or tyrosine residue; a mTOR polypeptide (SEQ ID NO: 2) having a phosphorylated serine, threonine 1 or tyrosine residue; a FKHR polypeptide (SEQ ID NO: 3) having a phosphorylated serine, threonine or tyrosine residue; a AKT polypeptide (SEQ ID NO: 4) having a phosphorylated serine, threonine or tyrosine residue; a ERK polypeptid
  • Yet another embodiment of the invention is a method to obtain information used to identify a therapeutic agent for treating glioblastoma in a human, the method comprising examining a cancer cell obtained from the human for the presence of: a S6 polypeptide (SEQ ID NO: 1) having a phosphorylated serine, threonine or tyrosine residue; a mTOR polypeptide (SEQ ID NO: 2) having a phosphorylated serine, threonine or tyrosine residue; a FKHR polypeptide (SEQ ID NO: 3) having a phosphorylated serine, threonine or tyrosine residue; a AKT polypeptide (SEQ ID NO: 4) having a phosphorylated serine, threonine or tyrosine residue; or decreased levels of expression of the PTEN polypeptide (SEQ ID NO: 5), wherein the presence of a phosphorylated S6, mTOR, FKHR or AKT polypeptide
  • the cancer cell is examined for the presence of a plurality of these characteristics.
  • the cancer cell is examined for the presence of a S6 polypeptide (SEQ ID NO: 1) having a phosphorylated serine, threonine or tyrosine residue and decreased levels of expression of the PTEN polypeptide (SEQ ID NO: 5).
  • the cancer cell is in a paraffin embedded biopsy sample.
  • embodiments of the invention typically utilize antibodies that specifically bind phosphorylated polypeptides, i.e. polypeptides having a phosphorylated serine, threonine or tyrosine residue.
  • the disclosure provides antibodies that bind to specific epitopes comprising a phosphorylated residue (e.g. serine at position 2481 in SEQ ID NO: 2).
  • a phosphorylated residue e.g. serine at position 2481 in SEQ ID NO: 2.
  • these phospho-specific antibodies can be used to examine the activation status of a pathway, where the activation is associated with phosphorylation of one or more specified residues.
  • the phosphorylation status and/ or expression levels of multiple members of a signalling pathway e.g. S6 and mTOR
  • Certain embodiments of the invention are used with formalin fixed, paraffin embedded biopsy samples.
  • the disclosure provided herein demonstrates that antibodies such as phospho-specific antibodies can be used with antigen samples processed in this manner.
  • the disclosure provided herein further demonstrates that the methods using these samples provide an accurate demonstration of the physiological status of the pathways in these samples. Consequently, the disclosure provided herein demonstrates how the methods of the invention are well suited for use with commonly available clinical samples.
  • the presence of a S6 polypeptide (SEQ ID NO: 1) having a phosphorylated serine, threonine or tyrosine residue is examined using an antibody that binds an epitope comprising a phosphorylated serine residue at position 235 in SEQ ID NO: 1.
  • the presence of a mTOR polypeptide (SEQ ID NO: 2) having a phosphorylated serine, threonine or tyrosine residue is examined using an antibody that binds an epitope comprising a phosphorylated serine residue at position 2481 in SEQ ID NO: 2.
  • the presence of a FKHR polypeptide (SEQ ID NO: 3) having a phosphorylated serine, threonine or tyrosine residue is examined using an antibody that binds an epitope comprising a phosphorylated threonine residue at position 24 in SEQ ID NO: 3.
  • the presence of a AKT polypeptide (SEQ ID NO: 4) having a phosphorylated serine, threonine or tyrosine residue is examined using an antibody that binds an epitope comprising a phosphorylated serine residue at position 473 in SEQ ID NO: 4.
  • additional polypeptide markers can also be examined.
  • additional markers include Ki-67 (SEQ ID NO: 9) and p-H3 histone H3 (SEQ ID NO: 10).
  • Yet another embodiment of the invention is a method of examining a mammalian cell for evidence of Akt pathway activation comprising examining the mammalian cell for the presence of: a S6 polypeptide (SEQ ID NO: 1) having a phosphorylated serine residue at position 235 in SEQ ID NO: 1; a mTOR polypeptide (SEQ ID NO: 2) having a phosphorylated serine residue at position 2481 in SEQ ID NO: 2; a FKHR polypeptide (SEQ ID NO: 3) having a phosphorylated threonine residue at position 24 in SEQ ID NO: 3; a AKT polypeptide (SEQ ID NO: 4) having a phosphorylated serine residue at position 473 in SEQ ID NO: 4; or decreased levels of expression of the PTEN polypeptide (SEQ ID NO: 5), wherein the presence of a phosphorylated S6, r ⁇ TOR, FKHR or AKT polypeptide, or decreased levels of expression of the PTEN polypeptide (
  • the mammalian cell is examined for the presence of a plurality of characteristics such as a S6 polypeptide (SEQ ID NO: 1) having a phosphorylated serine residue at position 235 in SEQ ID NO: 1 and decreased levels of expression of the PTEN polypeptide (SEQ ID NO: 5).
  • a S6 polypeptide SEQ ID NO: 1 having a phosphorylated serine residue at position 235 in SEQ ID NO: 1
  • decreased levels of expression of the PTEN polypeptide SEQ ID NO: 5
  • the mammalian cell is a cancer cell such as a cancer cell is of a glioblastoma lineage.
  • Certain embodiments of the invention comprise further methodological steps, for example using the results of the examination in a prognostic determination of tumor progression and/ or using the results of the examination to identify the presence of a glioblastoma characterized by a short time from initial diagnosis to patient death.
  • the further methodological steps include the step of using the results of the examination to identify a therapeutic agent for treating the glioblastoma such as the step of using the results of the examination to evaluate the effect of rapamycin on the glioblastoma cancer cell.
  • the mammalian cell is in a paraffin embedded biopsy sample.
  • a preferred embodiment of the invention is a method of examining a mammalian cell for evidence of Akt pathway activation comprising using a phospho- specific antibody to examine the cell for the presence of a phosphorylated protein in the mammalian cell selected from the group consisting of mTOR, FKHR and S6, wherein the presence of a phosphorylated mTOR, FKHR or S6 protein in the mammalian cell provides evidence of Akt pathway activation.
  • the cell is examined for the concurrent phosphorylation of mTOR, FKHR S6 proteins.
  • Such methods typically include an optional step of using a phospho-specific antibody to examine the cell for evidence of phosphorylation of a Akt protein in the mammalian cell.
  • the mammalian cell is typically a cancer cell that is present in a paraffin embedded biopsy sample.
  • the cancer cell is of the glioblastoma lineage.
  • Yet another embodiment of the invention is a method of examining a mammalian cell for evidence of Erk pathway activation comprising using a phospho- specific antibody to examine the cell for presence of phosphorylated p-44/42 MAP kinase proteins in the cells, wherein the presence of phosphorylated p-44/42 MAP kinase proteins in the mammalian cell provides evidence of Erk pathway activation.
  • the mammalian cell is present in a paraffin embedded biopsy sample obtained from an individual suspected of suffering from glioblastoma.
  • Another embodiment of the invention is a method of examining a tissue sample for the presence of mammalian glioblastoma cells having a phenotype characterized by a shorter time to tumor progression comprising using phospho-specific antibodies to examine the cell for the presence of phosphorylated mTOR, FKHR and S6 proteins in the cells, wherein the presence of a phosphorylated mTOR, FKHR and S6 proteins in the mammalian cell provides evidence of the phenotype.
  • a related embodiment of the invention is a method of examining a tissue sample for the presence of mammalian glioblastoma cells having a phenotype characterized by a short time from initial diagnosis to patient death comprising using phospho-specific antibodies to examine the cell for the presence of phosphorylated mTOR, FKHR and S6 proteins in the cells, wherein the presence of a phosphorylated mTOR, FKHR and S6 proteins in the mammalian cell provides evidence of the phenotype.
  • Another embodiment of the invention is a method of examining a tissue sample for the presence of mammalian glioblastoma cells having a phenotype characterized by a shorter time to tumor progression comprising using a phospho- specific antibody to examine the cell for the presence of phosphorylated Erk proteins in the cells, wherein the presence of a phosphorylated Erk proteins in the mammalian cell provides evidence of the phenotype.
  • a related embodiment of the invention is a method of examining a tissue sample for the presence of mammalian glioblastoma cells having a phenotype characterized by a short time from initial diagnosis to patient death comprising using phospho-specific antibodies to examine the cell for the presence of phosphorylated p-44/42 MAP kinase proteins in the cells, wherein the presence of a phosphorylated p-44/42 MAP kinase proteins in the mammalian cell provides evidence of the phenotype.
  • Yet another embodiment of the invention is a method of obtaining information useful for identifying an appropriate therapeutic agent to use to treat an individual suffering from glioblastoma comprising examining a tissue sample from the patient for the presence of glioblastoma cells having a phosphorylated protein selected from the group consisting of mTOR, FKHR and S6, wherein the presence of a phosphorylated mTOR, FKHR or S6 protein in the mammalian cell provides information useful for identifying an appropriate therapeutic agent to use to treat an individual suffering from glioblastoma.
  • the mammalian cell is examined for the presence of at least two and more preferably three phosphorylated proteins selected from the group consisting of mTOR, FKHR and S6.
  • the therapeutic agent is a kinase inhibitor of the Akt pathway.
  • Another embodiment of the invention is a method of obtaining information useful for identifying an appropriate therapeutic agent to use to treat an individual suffering from glioblastoma comprising examining a tissue sample from the patient for the presence of glioblastoma cells having phosphorylated Erk proteins, wherein the presence of phosphorylated Erk proteins in the mammalian cell provides information that can be used to identify an appropriate therapeutic agent to use to treat an individual suffering from glioblastoma.
  • Another embodiment of the invention is a method of examining a mammalian cell for evidence of Akt pathway activation comprising examining the cell for the expression of the EGFR and the EGFRvIII proteins, wherein the coexpression of the EGFR and the EGFRvIII proteins in the cell provides evidence of Akt pathway activation.
  • a related embodiment of the invention is a method of examining a mammalian cell for evidence of Erk pathway activation comprising examining the cell for the expression of the EGFR and the EGFRvIII proteins, wherein the coexpression of the EGFR and the EGFRvIII proteins in the cell provides evidence of Erk pathway activation.
  • Yet another embodiment of the invention is a method of examining a mammalian glioblastoma cell for evidence of Akt pathway activation, wherein the mammalian glioblastoma cell is obtained from a paraffin embedded biopsy sample, the method comprising examining the cell for decreased expression of the PTEN protein, wherein a decrease in the expression of the PTEN protein cell provides evidence of Akt pathway activation.
  • kits designed to facilitate the methods of the invention.
  • Such kits include instructions for using the elements therein according to the methods of the present invention.
  • kits can comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means can comprise one or more of the antibodies disclosed herein (an anti-S6 antibody for example) that is or can be detectably labeled with a marker.
  • kits utilizes immunological methods (e.g.
  • the kit can also have containers containing buffers for these methods and/ or containers comprising antibodies labelled with a reporter-means, such as a chromophore or radioactive molecule.
  • a reporter-means such as a chromophore or radioactive molecule.
  • additional reagents associated with these techniques can be further included in the kits.
  • an article of manufacture containing materials useful for the examination of the disorders described above comprises a container and a label.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container can hold a composition (e.g. an antibody composition) which is effective for examining mammalian cells (e.g. prostate cancer or glioblastoma cells).
  • the label on, or associated with, the container indicates that the composition is used for examining cellular polypeptides.
  • the article of manufacture may further comprise a second container comprising a buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a buffer such as phosphate-buffered saline, Ringer's solution and dextrose solution.
  • It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • kits comprising at least one antibody selected from the group consisting of: an antibody that binds a S6 polypeptide (SEQ ID NO: 1), wherein the S6 polypeptide epitope bound by the antibody comprises a phosphorylated serine, threonine or tyrosine residue; an antibody that binds a mTOR polypeptide (SEQ ID NO: 2), wherein the mTOR polypeptide epitope bound by the antibody comprises a phosphorylated serine, threonine or tyrosine residue; an antibody that binds a FKHR polypeptide (SEQ ID NO: 3), wherein the FKHR polypeptide epitope bound by the antibody comprises a phosphorylated serine, threonine or tyrosine residue; and an antibody that binds a AKT polypeptide (SEQ ID NO: 4), wherein the AKT polypeptide epitope bound by the antibody comprises a phosphorylated serine
  • kits of the invention further comprise an antibody that binds a PTEN polypeptide (SEQ ID NO: 5).
  • the kits of the invention can further include antibodies to additional polypeptides such as Ki-67 (SEQ ID NO: 9) and p-H3 histone H3 (SEQ ID NO: 10).
  • Another embodiment of the invention is a kit comprising an antibody capable of immunospecifically binding a phosphorylated protein in a mammalian cell selected from the group consisting of phosphorylated Akt, mTOR, FKHR and S6 proteins and instructions for using the antibody to examining the mammalian cell for evidence of Akt pathway activation.
  • the kit comprises different antibodies, each of which is capable of immunospecifically binding 2, 3 or 4 phosphorylated proteins in a mammalian cell selected from the group consisting of phosphorylated Akt, mTOR, FKHR and S6 proteins.
  • Another embodiment of the invention is a kit comprising an antibody capable of immunospecifically binding a phosphorylated p-44/42 MAP kinase proteins in a mammalian glioblastoma cell present in a paraffin embedded biopsy sample and instructions for using the antibody to examining the mammalian cell for evidence of Erk pathway activation.
  • kits for characterizing a mammalian prostate cancer or glioblastoma (GBM) tumor or cell comprising: an antibody that binds PTEN (SEQ ID NO: 5) and at least on of the following: an antibody that binds phosphorylated S6 ribosomal protein (SEQ ID NO: 1); an antibody that binds EFGR (SEQ ID NO: 7); an antibody that binds phosphorylated AKT (SEQ ID NO: 4); and/ or an antibody that binds phosphorylated ERK (SEQ ID NO: 8); and at least one secondary antibody that binds to the above noted primary antibodies.
  • the kit comprises a plurality of these antibodies.
  • the kit includes an antibody specific for S6 ribosomal protein (SEQ ID NO: 1) having a phosphorylated serine residue at position 235 in SEQ ID NO: 1; an antibody specific for AKT (SEQ ID NO: 4) having a phosphorylated serine residue at position 473 in SEQ ID NO: 4; or an antibody specific for ERK (SEQ ID NO: 8) having a phosphorylated threonine residue at position 202 or a phosphorylated tyrosine residue at position 204 in SEQ ID NO: 8.
  • SEQ ID NO: 1 an antibody specific for S6 ribosomal protein having a phosphorylated serine residue at position 235 in SEQ ID NO: 1
  • an antibody specific for AKT SEQ ID NO: 4
  • an antibody specific for ERK SEQ ID NO: 8 having a phosphorylated threonine residue at position 202 or a phosphorylated tyrosine residue at position 204 in SEQ ID NO: 8.
  • kits for characterizing a mammalian prostate cancer or glioma tumor or cell comprising: an antibody that binds PTEN (SEQ ID NO: 5); an antibody that binds phosphorylated S6 ribosomal polypeptide (SEQ ID NO: 1); an antibody that binds EFGR (SEQ ID NO: 7); an antibody that binds phosphorylated AKT (SEQ ID NO: 4); an antibody that binds phosphorylated ERK (SEQ ID NO: 8).
  • the kit further comprises a secondary antibody which binds to one of the primary antibodies directed to these polypeptides.
  • the kit comprises a plurality of antibodies such as an antibody specific for S6 ribosomal polypeptide (SEQ ID NO: 1) having a phosphorylated serine residue at position 235 in SEQ ID NO: 1, an antibody specific for AKT (SEQ ID NO: 4) having a phosphorylated serine residue at position 473 in SEQ ID NO: 4; or antibody specific for ERK having a phosphorylated threonine residue at position 202 and tyrosine 204 in SEQ ID NO: 8.
  • the kit further includes an antibody that binds Ki-67 polypeptide (SEQ ID NO: 9), p-H3 histone polypeptide (SEQ ID NO: 10) or caspase-3 polypeptide (SEQ ID NO: 11).
  • the methods of the present invention typically utilize antibodies directed to polypeptides in the PI3K/Akt pathway.
  • Illustrative antibody compositions useful in the present invention are anti-phosphoprotein antibodies characterized as containing antibody molecules that specifically immunoreacts with a phosphorylated form of a polypeptide associated with the PI3K/Akt pathway.
  • the polypeptide may be for example, S6, mTOR, FKHR, AKT or PTEN.
  • specifically immunoreacts it is meant that the antibody binds to the phosphorylated form of polypeptide (i.e. is phospho-specific) and does not bind to the unphosphorylated form of the same polypeptide. Consequently, the phosphorylation associated with pathway activation can be examined with such antibodies.
  • the antibodies of the invention can distinguish between the phosphorylated and unphosphorylated forms of a polypeptides associated with the PI3K/Akt pathway. Consequently, the phosphorylation associated with pathway activation can be examined with such antibodies.
  • the assays of the invention include immunohistochemical techniques using the antibodies disclosed herein. For example, a sample can be examined for the presence of a biochemical pathway associated phosphorylated polypeptide such as phosphorylated ERK by using an antibody that binds an epitope comprising a phosphorylated threonine residue at position 202 and tyrosine 204 in SEQ ID NO: 8.
  • the antibodies useful in the invention may comprise polyclonal antibodies, for example affinity purified polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/ or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include the appropriate polypeptide epitopes (e.g.
  • S6 polypeptide having a phosphorylated serine, threonine or tyrosine residue
  • a mTOR polypeptide having a phosphorylated serine, threonine or tyrosine residue
  • FKHR polypeptide SEQ ID NO: 3
  • a ERK polypeptide SEQ ID NO: 8
  • AKT polypeptide SEQ ID NO: 4
  • PTEN polypeptide PTEN polypeptide
  • the immunizing agent may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.
  • immunogenic proteins include but are not Limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • the antibodies may, alternatively, be monoclonal antibodies.
  • Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature. 256:495 (1975).
  • a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro.
  • the immunizing agent will typically include a phosphorylated 86, mTOR, FKHR, ERK or AKT polypeptide or PTEN polypeptide or a fusion protein thereof.
  • PBLs peripheral blood lymphocytes
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice. Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
  • HGPRT or HPRT hypoxanthine guanine phosphoribosyl transferase
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications. Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against phosphorylated S6, mTOR, FKHR, ERK or AKT polypeptides or PTEN and EGFR polypeptides.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem..
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567.
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the hybridoma cells of the invention serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then ttansfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • the DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4,816,567; Morrison et al., supra) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.
  • non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain.
  • the heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
  • the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art. [0103] Reactivity of antibodies with the cognate protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses. A antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme.
  • the invention provides assays for examining cellular pathways associated with disregukted cell growth. Certain embodiments of the invention include the steps of detecting the presence of phosphorylated S6, mTOR, FKHR, AKT or ERK polypeptides or PTEN and EGFR polypeptides in a tissue. Methods for detecting these polypeptides are well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. [0105] Typically the assays of the invention include immunohistochemical techniques.
  • Immunohistochemical techniques as used herein encompasses the use of reagents detecting cell specific markers, such reagents include, for example antibodies.
  • Antibodies including monoclonal antibodies, polyclonal antibodies and fragments thereof, are often used to identify proteins or polypeptides of interest in a sample.
  • a number of techniques are utilized to label objects of interest according to immunohistochemical techniques. Such techniques are discussed in Current Protocols in Molecular Biology, Unit 14 et seq., eds. Ausubel, et al., John Wiley & Sons, 1995, the disclosure of which is incorporated herein by reference. Typical protocols include staining a paraffin embedded tissue section prepared according to a conventional procedure (see, e.g. U.S. Patent No. 6,631,203).
  • Certain embodiments of the invention include tunel assays a markers of apoptosis.
  • a TUNEL, assay is performed essentially as follows: the percentage of apoptotic cells are detected by the APO-BRDU terminal deoxynucleotidyl transferase (TdT) -mediated dUTP-biotin nick end-labeling assay (see, e.g. Gavrieli, et al., J. Cell Biol. 119: 493-501) according to manufacturer's instructions (see, e.g. Phoenix Flow Systems, Phoenix, AZ).
  • TUNEL assays useful in methods of the invention see, e.g.
  • caspase-3 assays include caspase-3 assays.
  • the caspase-3 assay measures the activation of caspase-3 enzyme, a critical early event of apoptosis induced death (see, e.g. U.S. Patent Application No. 20020159996 and U.S. Patent No. 6,346,607).
  • TUNEL assays useful in methods of the invention see, e.g. Duan et al., J Pathol. 2003 Feb;199(2):221-8; and Walker et al., J Pathol. 2001 Oct;195(3):275-6.
  • various publications are referenced. The disclosures of these publications are hereby incorporated by reference herein in their entireties.
  • Examples 1-8 provide illustrative disclosure showing embodiments of the invention used to examine glioma cells.
  • Example 9 provides an illustrative disclosure showing embodiments of the invention used to examine prostate cancer cells.
  • EXAMPLE 1 Patient selection and construction of Tissue Microarray:
  • Font tumors had sufficient material on the tissue array for analysis of PTEN, EGFR and EGFRvIII, but lacked sufficient material for analysis of p-Akt, p-mTOR, p- S6, p-FKHR and ⁇ -Erk.
  • Heat-induced antigen retrieval was used as follows: for p-Erk, p-Akt, p- mTOR, p-FKHR/FKHRLl and p-s6, 0.01 M citrate buffer, pH 6 for 25 minutes in a pressure cooker; for PTEN, 0.01M citrate buffer, pH 6 for 16 minutes in a microwave oven; EGFR, pronase (0.03 g/ml Of 0.05 M Tris buffer, pH 7.4) at 37°C for 8 minutes and for EGFRvIII, 0.01 M citrate buffer, pH 6 for 25 minutes in a vegetable steamer. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol.
  • PTEN - PTEN staining was scored according to a previously established scale of 0-2, in which the vascular endothelium (score of 2) serves as an internal control (see, e.g., Perren et al., Am J Pathol. 157: 1097-103., 2000; Perren et al, Am J Pathol. 155: 1253-60., 1999; Zhou et al., Am J Pathol. 161: 439-47., 2002; Gimm et al., Am J Pathol. 156: 1693-700., 2000).
  • Tumor cells are graded as 2 if their staining intensity is equal to that of the vascular endothelium, 1 if it is diminished relative to the endothelium, and 0 if it is undetectable in the tumor cells and present in the vascular endothelium (see, e.g., Zhou et al., AmJ Pathol. 161: 439-47., 2002).
  • This scoring system has been shown to be highly consistent between different cancer cell types, including breast (see, e.g., Perren et al., AmJ Pathol. 155: 1253-60., 1999), ovarian (see, e.g., Mutter et al., Cancer Res.
  • pancreas see, e.g., Perren et al., Am J Pathol. 157: 1097-103., 2000
  • colon see, e.g., Zhou et al., Am J Pathol. 161: 439-47., 2002.
  • Two Neuropathologists scored the tumors independently. In addition, tumors were scored by one of the Neuropathologists on two independent occasions. Both the inter- rater, and the intra-rater agreement were greater than 90%.
  • EGFR and EGFRPIII - Tumors demonstrating strong EGFR immunopositivity in greater than 20% of tumor cells were considered to be positive (see, e.g., Liotta et al., Jama. 286: 2211-4., 2001); tumors demonstrating at least focal moderate to strong immunoreactivity for EGFRvIII were considered positive, as previously reported (see, e.g., Choe et al., Clin Cancer Res. 8: 2894-901., 2002).
  • the inter-rater and intra-rater agreement for EGFR and EGFRvIII were > 90%.
  • S6 is phosphorylated by p70 S6 kinase, which is itself a target of Akt (see, e.g., Blume-Jensen et al., Nature. 411: 355-365., 2001).
  • Akt activation was also correlated with strong S6 phosphorylation (2+) (p ⁇ O.OOl), although weaker S6 phosphorylation (1+) was also detected in Akt- negative tumors (Table 3).
  • EXAMPLE 6 Akt pathway activation in GBMs lacking PTEN protein loss: assessment of EGFR/EGFRvIII-mediated signaling
  • PTEN loss did not appear to be the only route to Akt activation; expression of p-Akt and downstream effectors p-mTOR, p-FKHR and p-S6 was also detected in 28% of GBMs with no immunohistochemical PTEN loss (Table 2). Because the PI3'K/Akt pathway can be activated by EGFR signaling, we analyzed EGFR and EGFRvIII expression and assessed their association with PI3'K/Akt pathway activation in the setting of normal PTEN immunohistochemical staining. EGFR immunopositivity was detected in 60% of GBMs (Fig. 2), in line with previous reports (see, e.g., Smith et al., J Natl Cancer Inst.
  • This finding may reflect a contribution from additional inputs downstream of Akt, such as Erk-mediated activation of S6 kinase and nutrient-mediated activation of mTOR.
  • this panel of three phospho-specific antibodies may be a more sensitive method to detect Akt pathway activation than a single phospho-Akt antibody alone.
  • Erk activation was also significantly associated with more rapid progression and diminished overall survival in this subset of primary GBM patients ( ⁇ 0.04) (Table 5).
  • Figures 3A and 3B provide an illustration of the interaction between members of the PI3K/Akt pathway and kinase inhibitors.
  • Figure 3A shows that rapamycin inhibits S6 phosphorylation in glioblastoma in vivo.
  • Figure 3A provides data from an analysis of a cohort of patients on a rapamycin clinical trial. This data shows that a substantial reduction in
  • Control patients showed a uniformly high level of S6 phosphorylation.
  • This data provides evidence that rapamycin inhibited mTOR signaling at the level of S6 phosphorylation in the majority of glioblastoma patients.
  • this data illustrates how the detection of pathway activation by immunohistochemistry (IHC) correlates with detection by western blotting.
  • IHC immunohistochemistry
  • FIG. 3B shows that the rapamycin-mediated inhibition of S6 phosphorylation correlates with diminished tumor proliferation.
  • Ki-67 a marker of cellular proliferation was used to assess whether rapamycin-mediated inhibition of S6 had an effect on tumor growth. This data provides evidence that the rapamycin-mediated inhibition of mTOR signaling at the level of S6 phosphorylation correlated with diminished tumor cell proliferation.
  • EXAMPLE 9 Antibody based profiling of the PI3K pathway in clinical prostate cancer.
  • this Example illustrates the detection of activated signaling pathways in situ on formalin fixed, paraffin embedded tissue, analogous to detecting Her-2/Neu amplification by immunohistochemistry in breast cancers.
  • the target mTOR
  • mTOR is a kinase
  • PTEN loss was highly correlated with AKT activation, and AKT activation was correlated with upregulation of p-S6.
  • Human prostate cancer tissue microarray [0128] We constructed a tissue microarray from formalin fixed, paraffin embedded archival tissue blocks, from 133 radical prostatectomies, performed for prostate cancer. The distribution of Gleason scores and Pathological TNM stage were as follows: 79 cases had Gleason score 5-6; 37 cases Gleason score 7 and 9 cases Gleason score 8-10. Eighty four cases were organ confined (i.e. Stage 2), 34 were Stage 3 and 13 cases were Stage 4. The array was designed so that three cotes were taken from tumor bearing tissue and one core from morphologically benign tissue (only areas containing normal prostate glands and stroma were sampled; atrophic and hyperplastic glands were not included in the array construction or in the evaluation), from the same patient. These cores were placed adjacent to each other on the tissue array, enabling comparison of antibody staining pattern and intensity between benign and cancer tissue from the same patient.
  • CeUs (LNCaP, DU 145, 1 HT 129) were grown in culture (DMEM supplemented with 10% FBS, L-Glutamine, Penicillin and Streptomycin) and exposed to vehicle, the PDK inhibitor LY294002 (30 ⁇ M), the mTOR inhibitor, CCI-779 (2OnM) for 18 hours or the MEK inhibitor, UO126 (20 ⁇ M) combined with lOOng/ml of EGF for 20 minutes. Cell pellets were prepared, formalin fixed and embedded in paraffin, then cut for immunohistochemical staining with phospho-AKT, -S6, -FKHR and -ERK antibodies.
  • This antibody recognizes a 99 amino acid epitope at the C-terminus of PTEN) and polyclonal phosphorylation-specific antibodies to p-AKT (Ser 473), p-FKHR which recognizes phosphorylated forms of the forkhead family of transcription factors FKHR (FOXO 1), FKHRLl (FOXO 3a) and AFX (FOXO 4), p-S6 Ribosomal protein (Ser 235/236), p- ERK (Thr-202/Tyr-204) at 1:100 (Cell Signaling Technology, Beverly, MA). This was followed by application of biotinylated secondary antibodies (Vector) at 1:1000 dilution for 30 minutes and Avidin-biotin complex (Elite ABC; Vector).
  • p-AKT Ser 473
  • p-FKHR which recognizes phosphorylated forms of the forkhead family of transcription factors FKHR (FOXO 1), FKHRLl (FOXO 3a) and AFX (FO
  • Negative control slides received normal mouse serum (DAKO) as the primary antibody.
  • Diaminobenzidine tetrahydrochloride (DAB) was used as the enzyme substrate to visualize specific antibody localization for PTEN and p-AKT; Vector NovaRed (Vector) for p-FKHR, p-S6 and p- ERK.
  • Slides were counterstained with Harris hematoxylin.
  • PTEN staining was scored according to a previously established scale of 0-2, which has been shown to be highly consistent (Choe et al., Cancer Res, 63: 2742- 2746, 2003; Gimm et al., Am J Pathol, 156: 1693-1700, 2000; Perren et al., Am J Pathol, 155: 1253-1260, 1999; Zhou et al., AmJ Pathol, 157: 1123-1128, 2000).
  • Tumor cells are graded as 2 if their staining intensity is equal to that of the adjacent benign cells, graded as 1 if their staining was diminished relative to the benign cells and 0 if staining intensity is undetectable in the tumor cells and is present in the benign cells.
  • immunohistochemical assays will identify loss of protein expression in tumors with homozygous deletions, nonsense mutations, certain internal deletions, promoter methylation and finally posttranscriptional modifications.
  • MDS is an unsupervised data analysis method that allows examination of potential relationships between variables without assuming previous knowledge of their interaction.
  • the signaling molecules are plotted in two dimensions and the distance between two molecules provide a measure of their inter-relatedness (Venables et al, Modern Applied Statistics with S-Plus. New York: Springer, 1999).
  • This plot is a graphical representation of the correlations found in Table 7. Loss of PTEN protein expression and AKT activation are closely related, as shown by the short distance between them. In contrast, p-ERK is distant from PTEN and p-AKT ( Figure 7).
  • PTEN loss when examined by Pearson and Multivariate analysis, is significantly associated with phosphorylation of AKT. ConceptuaUy, it may be that PTEN score 1 may reflect decreased (but not absent) gene activity.
  • p- FKHR scored the lowest in the intra and inter-observer correlation percentiles, suggesting that subtle differences in staining intensity may lead to variations ' in the scoring protocol.
  • p-ERK is not correlated with any molecules in this pathway, consistent with in vitro data that places the MAPK pathway paraUel to, rather than epistatic to the PI3K cascade.
  • Table 6 provides the sequences, accession numbers and illustrative references for the well known polypeptides discussed herein. In certain sequences in this Table, illustrative residues that are typically phosphorylated during pathway signalling are shown in boldface type.
  • PTEN (NP 000305, gi;4506249) 403 amino acids See, e.g. Li et al. , Science 275 (5308) , 1943-1947 (1997)
  • EGFR (NP 005219 # gi;29725609) 1210 amino acids See, e.g. Tarn et al. , Nature 309 (5967), 418-425 (1984)
  • PTEN p-AKT 0.28 a (0.14-0.58) 0.0005 p-S6 0.5 (0.30-1.04) 0.065 p-FKHR 0.99 (0.51-2.00) 0.97 p-AKT p-S6 2.17 (1.09-4.32) 0.028 p-FKHR 0.98 (0.49-1.95) 0.95
  • Odds of a tumor being PTEN deficient if it is p-AKT positive are 3.57 times (1/0.28) greater than if it is p-AKT negative

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des procédés permettant d'examiner et/ou de quantifier les voies biochimiques PI3K/Akt et Ras/MAPK qui sont dérégulées dans des pathologies de type cancers de la prostate et gliomes. L'invention concerne également des réactifs et des kits conçus pour mettre en oeuvre lesdits procédés.
PCT/US2004/037288 2003-05-11 2004-11-08 Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate Ceased WO2006052249A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US2004/037288 WO2006052249A1 (fr) 2004-11-08 2004-11-08 Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate
US11/667,430 US20080299589A1 (en) 2003-05-11 2004-11-08 Methods and Materials for Examining the P13k/Akt Pathway in Gliomas and Prostate Cancers

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2004/037288 WO2006052249A1 (fr) 2004-11-08 2004-11-08 Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate

Publications (1)

Publication Number Publication Date
WO2006052249A1 true WO2006052249A1 (fr) 2006-05-18

Family

ID=34959462

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2004/037288 Ceased WO2006052249A1 (fr) 2003-05-11 2004-11-08 Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate

Country Status (1)

Country Link
WO (1) WO2006052249A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008132494A1 (fr) * 2007-05-01 2008-11-06 Ludwig Institute Of Cancer Research Variante tronquée de la mtor (cible de la rapamycine chez les mammifères)
WO2008125633A3 (fr) * 2007-04-13 2009-05-28 Rikshospitalet Radiumhospitale Traitement et diagnostic du cancer métastatique de la prostate par des inhibiteurs de récepteur de facteur de croissance epidermique (egfr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087761A2 (fr) * 2002-04-05 2003-10-23 Cell Signaling Technology, Inc. Profilage de molecules d'une maladie et reponse therapeutique utilisant des anticorps phospho-specifiques
WO2004044218A2 (fr) * 2002-11-05 2004-05-27 The Regents Of The University Of California Procedes, substances et materiel permettant d'examiner des voies associees a la progression de glioblastome

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003087761A2 (fr) * 2002-04-05 2003-10-23 Cell Signaling Technology, Inc. Profilage de molecules d'une maladie et reponse therapeutique utilisant des anticorps phospho-specifiques
WO2004044218A2 (fr) * 2002-11-05 2004-05-27 The Regents Of The University Of California Procedes, substances et materiel permettant d'examiner des voies associees a la progression de glioblastome

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PANIGRAHI A R ET AL: "The role of PTEN and its signalling pathways, including AKT, in breast cancer; an assessment of relationships with other prognostic factors and with outcome", JOURNAL OF PATHOLOGY, vol. 204, no. 1, September 2004 (2004-09-01), pages 93 - 100, XP008050606, ISSN: 0022-3417 *
XU GUANG ET AL: "Pharmacogenomic profiling of the PI3K/PTEN-AKT-mTOR pathway in common human tumors.", INTERNATIONAL JOURNAL OF ONCOLOGY. APR 2004, vol. 24, no. 4, April 2004 (2004-04-01), pages 893 - 900, XP008050608, ISSN: 1019-6439 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008125633A3 (fr) * 2007-04-13 2009-05-28 Rikshospitalet Radiumhospitale Traitement et diagnostic du cancer métastatique de la prostate par des inhibiteurs de récepteur de facteur de croissance epidermique (egfr)
WO2008132494A1 (fr) * 2007-05-01 2008-11-06 Ludwig Institute Of Cancer Research Variante tronquée de la mtor (cible de la rapamycine chez les mammifères)
CN101939332A (zh) * 2007-05-01 2011-01-05 路德维格癌症研究所 哺乳动物雷帕霉素靶标(mTOR)蛋白的截短变体
US8669347B2 (en) 2007-05-01 2014-03-11 Ucl Business Plc Truncated variant of the mammalian target for rapamycin (MTOR) protein
CN101939332B (zh) * 2007-05-01 2014-03-19 路德维格癌症研究所 哺乳动物雷帕霉素靶标(mTOR)蛋白的截短变体

Similar Documents

Publication Publication Date Title
US20040106141A1 (en) Methods and materials for examining pathways associated with glioblastoma progression
Jensen et al. PIK3CA mutations, PTEN, and pHER2 expression and impact on outcome in HER2-positive early-stage breast cancer patients treated with adjuvant chemotherapy and trastuzumab
Ayala et al. The prolyl isomerase Pin1 is a novel prognostic marker in human prostate cancer
Khan et al. Early diagnostic value of survivin and its alternative splice variants in breast cancer
Gluz et al. Triple-negative high-risk breast cancer derives particular benefit from dose intensification of adjuvant chemotherapy: results of WSG AM-01 trial
Goldstein et al. Epidermal growth factor receptor immunohistochemical reactivity in patients with American Joint Committee on Cancer Stage IV colon adenocarcinoma: implications for a standardized scoring system
Tan et al. HER-2/neu Protein Expression and Gene Alteration in Stage I-IIIA Non–Small-Cell Lung Cancer: A Study of 140 Cases Using a Combination of High Throughput Tissue Microarray, Immunohistochemistry, and Fluorescent In Situ Hybridization
Wu et al. Ano1/TMEM16A overexpression is associated with good prognosis in PR-positive or HER2-negative breast cancer patients following tamoxifen treatment
Dimou et al. Measurement of aldehyde dehydrogenase 1 expression defines a group with better prognosis in patients with non-small cell lung cancer
Umemura et al. Estrogen receptor-negative and human epidermal growth factor receptor 2-negative breast cancer tissue have the highest Ki-67 labeling index and EGFR expression: gene amplification does not contribute to EGFR expression
EP1368630A2 (fr) Proteines ou acides nucleiques pin1 utilises comme marqueur de croissance cellulaire anormale
Murakawa et al. Expression of KIT, EGFR, HER‐2 and tyrosine phosphorylation in undifferentiated thyroid carcinoma: implication for a new therapeutic approach
Choi et al. Immunohistochemical biomarkers in patients with early-onset breast carcinoma by tissue microarray
US20110045459A1 (en) Molecular determinants of EGFR kinase inhibitor response in glioblastoma
Bai et al. Comparison of HER2 and phospho-HER2 expression between biopsy and resected breast cancer specimens using a quantitative assessment method
O'Callaghan K-ras mutations and benefit from cetuximab in advanced colorectal cancer
Da Silva et al. Pilot assessment of vascular endothelial growth factor receptors and trafficking pathways in recurrent and metastatic canine subcutaneous mast cell tumours
WO2007105110A2 (fr) Expression d'ercc1 dans la prédiction de réponse pour la chimiothérapie du cancer
Wilkinson et al. Localized high-risk prostate cancer harbors an androgen receptor low subpopulation susceptible to HER2 inhibition
WO2006052249A1 (fr) Procedes impliquant la voie de signalisation pi3k/akt dans des gliomes et des cancers de la prostate
Kanjer et al. Treatment response to preoperative anthracycline-based chemotherapy in locally advanced breast cancer: the relevance of proliferation and apoptosis rates
Seo et al. Differential Clinical Significance of Neurotrophin-3 Expression according to MYCN Amplification and TrkC Expression in Neuroblastoma
US20120134995A1 (en) Method for predicting the therapeutic responseiveness of a patient to a medical treatment with an egfr inhibitor
US20080299589A1 (en) Methods and Materials for Examining the P13k/Akt Pathway in Gliomas and Prostate Cancers
US8465936B2 (en) Method for determining the sensitivity of patients suffering from a cancer disease to biological therapy

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase

Ref document number: 04822417

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 11667430

Country of ref document: US