WO2017165663A1 - Biomarqueurs pour un cancer lié à l'activité de la voie hedgehog et utilisations associées de ces biomarqueurs - Google Patents

Biomarqueurs pour un cancer lié à l'activité de la voie hedgehog et utilisations associées de ces biomarqueurs Download PDF

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WO2017165663A1
WO2017165663A1 PCT/US2017/023839 US2017023839W WO2017165663A1 WO 2017165663 A1 WO2017165663 A1 WO 2017165663A1 US 2017023839 W US2017023839 W US 2017023839W WO 2017165663 A1 WO2017165663 A1 WO 2017165663A1
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cancer cells
cilia
antibodies against
expression
inhibitor
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Kimberly MCDERMOTT
Nadia Bassam HASSOUNAH
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University of Arizona
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to biomarkers for detection and characterization of cancer cells associated with hedgehog pathway activity (e.g., non-small cell lung cancer) and related uses thereof.
  • hedgehog pathway activity e.g., non-small cell lung cancer
  • Lung cancer is a highly recalcitrant cancer and remains the leading cause of cancer- related deaths worldwide with non-small cell lung cancer (NSCLC) accounting for the maj ority (-80%) of all cases.
  • NSCLC non-small cell lung cancer
  • the hedgehog (Hh) pathway drives tumor cell survival in many cancers, including
  • Inhibitors that target the Hh pathway such as vismodegib (2-Chloro-N-(4-chloro- 3-pyridin-2-ylphenyl)-4-methylsulfonylbenzamide) have shown impressive disease regression in basal cell carcinoma of the skin and medulloblastoma.
  • drug responsiveness varies dramatically between patients from complete remission in some patients and no response in others.
  • Frustratingly, the use of tumor markers of increased Hh activity was not found to be predictive of patient response to Hh-targeted treatment. This failure of the biological correlate between the target and response suggests a significant and fundamental gap in understanding of the molecular mechanisms that regulate Hh- targeted drug efficacy. Addressing this gap is critical and of high potential impact in the effort to better guide the use of the Hh inhibitors for cancer treatment.
  • the present invention addresses such needs.
  • Hh pathway can be activated via cilia-dependent or cilia- independent mechanisms (see, e.g., Hassounah, ⁇ . ⁇ ., et al, 2012 Clin Cancer Res 18, 2429-2435).
  • Hedgehog ligands are a family of secreted proteins that include Sonic Hh (Shh), Indian Hh (Ihh), and Desert Hh (Dhh). These Hh ligands activate the downstream Gli family of transcription factors that translocate into the nucleus to activate Hh target genes (this constitutes increased Hh signaling).
  • Cilia-dependent activation of the Hh pathway occurs because the cilium itself is a subcellular compartment in which key Hh pathway components including Gli proteins are brought together and processed
  • cilia-independent Hh signaling occurs when cilia are lost (therefore loss of Gli ⁇ which inhibits transcriptional activation of Hh genes). This leaves promoters of Hh genes vulnerable to activation by oncogenic transcription factors including Myc (Fig. IB). Pharmacological inhibition of the Hh pathway in cilia-dependent cancers would therefore be mechanistically different than that of cilia-independent cancers (Fig. 2).
  • the present invention provides methods for selecting an individual having cancer cells associated with Hh pathway activity for treatment with a Hh inhibitor, comprising
  • the present invention provides methods for treating a human subject having cancer associated with Hh pathway activity, comprising the steps of a) performing a nucleic acid-based detection assay to determine a gene expression profile of one or more biomarker genes in a sample comprising cancer cells obtained from the human subject; b) comparing the determined gene expression profile with a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression; c) determining that the human subject is responsive or unresponsive to a Hh inhibitor treatment based on such a comparison; and d) administering an effective amount of a Hh inhibitor to the human subject having a determined gene expression profile that is consistent with the reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • the present invention provides methods for determining the presence or absence of primary cilia expression in cancer cells associated with Hh pathway activity, comprising a) determining a gene expression profile of one or more biomarker genes within a sample comprising such cancer cells, b) comparing the determined gene expression profile with a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression, and c) identifying such cancer cells as having primary cilia expression if the determined gene expression profile is consistent with the reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • the one or more biomarker genes include one or more genes involved in ciliogenesis. In some embodiments, the one or more biomarker genes include one or more genes involved in cilia function. In some embodiments, the one or more biomarker genes include one or more genes involved in downstream pathway activity related to ciliogenesis and/or cilia function. In some embodiments, the one or more biomarker genes include one or more genes associated with cilia disease. In some embodiments, the one or more biomarker genes include one or more genes associated with Hh pathway related activity. In some embodiments, the one or more biomarker genes include one or more genes involved in downstream pathway activity realted to ciliogenesis and/or cilia function and one or more genes associated with Hh pathway related activity.
  • the one or more biomarker genes are selected from the biomarker genes recited in Figs. 7, 8, and/or 9 (e.g., ABHD4, ACSS2, ARL14, BLOC1S2, C17orf91, CDK 1A, CLIC3, CLIP3, CYP4F11, DDIT3, DDIT4, GABARAPL1, GCNT2, GDF15, GPX3, GULP1, HKDC1, IDI1, IL1RN, INSIG1, KIAA1370, LCN2, LCP1, LPIN1, MVD, NUPR1, OSGIN1, PANX2, PCSK9, RBCK1, SC4MOL, SCD, SDCBP2, STOX2, TIMP4, TP53INP1, TRIM31, UBC, YPEL5, ARHGAP11A, ASF1B, AURKB, BIRC5, BLM, C15orf42, C16orf59, CASC5, CCNA2, CCNE2, CDC20, CDC6,
  • TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS1, PHKHD1, RABL5, HSPB11, PKDl, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCBl see, e.g., Example VI.
  • Such methods are not limited to incorporating a specific number of such biomarker genes in determining the gene expression profile.
  • 1 biomarker gene is utilized.
  • 5 biomarker genes are utilized in determining the gene expression profile.
  • more than 10 e.g., 10, 11, 12, 15, 20, 25, 50, 100, 1000, etc. are utilized in determining the gene expression profile.
  • determining the gene expression profile involves quantifying mRNA expression related to the specific biomarker genes. In some embodiments, determining the gene expression profile involves quantifying protein expression related to the specific biomarker genes. In some embodiments, determining the gene expression profile involves quantifying mRNA expression alnd protein expression related to the specific biomarker genes .
  • the cancer having cells associated with Hh pathway activity is selected from NSCLC, bladder cancer, basal cell carcinoma, medulloblastoma, colon cancer, breast cancer, and pancreatic cancer.
  • Such methods are not limited to a particular type of Hh inhibitor.
  • the Hh inhibitor is a cilia dependent Hh inhibitor.
  • the Hh inhibitor is Taladegib (LY2940680). In some embodiments, the Hh inhibitor is vismodegib. In some embodiments, the Hh inhibitor is selected from Taladegib (LY2940680), sulforaphane, vismodegib, TAK-441, itraconazole, and erismodegib.
  • the Hh inhibitor is selected from Hh ligand inhibitors (e.g.,
  • Hh inhibitor is described and disclosed in U.S. Patent 7,230,004, U.S. Patent Application Publication No. 2008/0293754, U.S. Patent Application Publication No. 2008/0287420, U.S. Patent Application Publication No. 2008/0293755.
  • Hh inhibitors include, but are not limited to, those described in U.S. Patent Application Publication Nos. US 2002/0006931, US 2007/0021493 and US 2007/0060546, and International Application Publication Nos. WO 2001/19800, WO 2001/26644, WO 2001/27135, WO 2001/49279, WO 2001/74344, WO 2003/011219, WO 2003/088970, WO 2004/020599, WO 2005/013800, WO 2005/033288, WO 2005/032343, WO 2005/042700, WO 2006/028958, WO 2006/050351, WO 2006/078283, WO 2001/19800, WO 2001/26644, WO 2001/27135, WO 2001/49279, WO 2001/74344, WO 2003/011219, WO 2003/088970, WO 2004/020599, WO 2005/013800, WO 2005/033288, WO 2005/032343, WO 2005/04
  • 2007/054623 WO 2007/059157, WO 2007/120827, WO 2007/131201, WO 2008/070357, WO 2008/110611, WO 2008/112913, and WO 2008/131354.
  • Hh inhibitors include, but are not limited to, BMS-
  • 833923 also known as XL139 (also known as XL139) (see, e.g., Siu L. et al, J. Clin. Oncol. 2010; 28: 15s (suppl; abstr 2501)); LDE-225 (see, e.g., Pan S. et al, ACS Med. Chem. Lett., 2010; 1(3): 130-134); LEQ-506 (see, e.g., National Institute of Health Clinical Trial Identifier No. NCT01106508); PF-04449913 (see, e.g., National Institute of Health Clinical Trial Identifier No.
  • NCT00953758 Hh pathway antagonists disclosed in U.S. Patent Application Publication No. 2010/0286114; SMOi2-17 (see, e.g., U.S. Patent Application Publication No.
  • the reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression is generated from one or more subjects (e.g., 1, 5, 10, 100, 1000, 10,000, etc) diagnosed as having a cancer (e.g., NSCLC, bladder cancer, basal cell carcinoma, medulloblastoma, colon cancer, breast cancer, and pancreatic cancer) having cells (e.g., NSCLC cells, bladder cancer cells, basal cell carcinoma cells, medulloblastoma cells, colon cancer cells, breast cancer cells, and pancreatic cancer cells) associated with Hh pathway activity and primary cilia expression.
  • a cancer e.g., NSCLC, bladder cancer cells, basal cell carcinoma cells, medulloblastoma cells, colon cancer cells, breast cancer cells, and pancreatic cancer cells
  • the reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression is generated as described in Example VI.
  • such methods can be used to predict the likelihood of a patient's ability to effectively respond to a Hh inhibitor based treatment for a cancer associated with Hh pathway activity.
  • such methods can be used for screening the effectiveness of new Hh inhibiting drugs in the treatment of a cancer associated with Hh pathway activity.
  • such methods can be used for identifying new Hh inhibiting drugs in the treatment of a cancer associated with Hh pathway activity.
  • the present invention provides methods for determining the presence or absence of primary cilia expression in cancer cells associated with Hh pathway activity, comprising obtaining a biological sample comprising cancer cells, exposing the cancer cells to antibodies against a ciliary axoneme, antibodies against a cilia associated centrosome (e.g., a centrosome attached to a ciliar axoneme), antibodies against markers for activated Hh signaling, and three different fluorescently conjugated secondary antibodies to distinguish between the antibodies against a ciliary axoneme, the antibodies against a cilia associated centrosome, and the antibodies against markers for activated Hh signaling, and detecting the presence or absence of binding between the antibodies against a ciliary axoneme, detecting the presence or absence of binding between antibodies against a cilia associated centrosome, and detecting the presence or absence of binding between antibodies against markers for activated Hh signaling, wherein a detected presence of binding between the antibodies against a ciliary axoneme, the antibodies against a cili
  • Such methods for determining the presence or absence of primary cilia expression in cancer cells associated with Hh pathway activity are not limited to utilizing specific antibodies.
  • the antibody against a ciliary axoneme i s anti-acetylated- tubulin.
  • the antibodies against a cilia associated centrosome is anti- gamma (Y)-tubulin.
  • the antibodies against markers for activated Hh signalling is anti-Ptchl antibody and/or anti-Glil antibody and/or anti-SMO antibody.
  • the present invention provides methods for selecting an individual having cancer cells associated with Hh pathway activity for treatment with a Hh inhibitor, comprising obtaining a biological sample from the individual, wherein the biological sample comprises cancer cells, detecting the presence or absence of primary cilia expression within the cancer cells, and administering to the individual a therapeutically effective amount of a cilia-dependent Hh inhibitor if primary cilia expression is detected in the cancer cells, or not administering to the individual a therapeutically effective amount of a cilia-dependent Hh inhibitor if primary cilia expression is not detected in the cancer cells.
  • the present invention provides methods for treating a human subject having cancer cells associated with Hh pathway activity, comprising the steps of obtaining a biological sample from the human subject, wherein the biological sample comprises cancer cells, detecting the presence or absence of primary cilia expression within the cancer cells, and administering to the individual a therapeutically effective amount of a cilia-dependent Hh inhibitor if primary cilia expression is detected in the cancer cells, or not administering to the individual a therapeutically effective amount of a cilia-dependent Hh inhibitor if primary cilia expression is not detected in the cancer cells.
  • the cancer cells associated with Hh pathway activity are selected from NSCLC cells, bladder cancer cells, basal cell carcinoma cells, medulloblastoma cells, colon cancer cells, breast cancer cells, and pancreatic cancer cells.
  • the cilia-dependent Hh inhibitor is selected from Taladegib (LY2940680), sulforaphane, vismodegib, TAK-441, itraconazole, erismodegib, 5E1, robotnikinin, IPI-926, HPI-2, HPI-3, HPI-4, and arsenic trioxide.
  • FIG. 1 demonstrates that primary cilia are both positive and negative regulators of the Hh pathway.
  • FIG. 2A-D shows cilia-dependent activation of the Hh pathway.
  • FIG. 3 shows the presence of cilia on basal cell carcinoma is predictive of responsiveness to GDC-0449 treatment.
  • A Human tissues were stained for primary cilia (example marked with white arrow) using antibodies that recognize Acetylated-Tubulin (grey-scaled red, ciliary axoneme) and gamma-tubulin (grey-scaled green, centrosome). Staining for Epcam (grey-scaled white) was used to mark epithelia and cancer cells.
  • FIG. 4 shows that primary cilia are expressed on NSCLC cells.
  • a Human NSCLC tissues and B. human NSCLC cell A549 were stained for primary cilia (grey-scaled white arrows) using antibodies that recongnize Acetylated -Tubulin (grey-scaled red, ciliary axoneme) and gamma-Tubulin (grey-scaled green, centrosome). Dashed box shows highly ciliated cancer cells.
  • FIG. 5 shows NSCLC patient-derived xenografts have differential expression of primary cilia.
  • LAC PDX from patient #1 (Cilia-High) and patient #2 (Cilia-Low) were stained for markers of primary cilia and assigned a cilia score based on percentage of ciliated cells normalized to cell cycle (Ki67 index).
  • FIG. 6 shows Hh signature correlates with high ciliogenesis signature in a subset of
  • FIG. 7 shows a list of genes upregulated when the cancer cells are treated with GANT61 (an inhibitor of GLI1 and GLI2, both of which are transcriptional regulators of Hh signaling). Therefore, these genes would have downregulated expression when Hh is ON.
  • FIG. 8 shows a list of genes downregulated when the cancer cells are treated with
  • GANT61 Inhibitor an inhibitor of GLI1 and GLI2, both of which are transcriptional regulators of Hh signaling. Therefore, these genes would have upregulated expression when Hh is ON.
  • FIG. 9 shows a list of genes that are associated with primary cilia in one or more of the following ways: 1) required for ciliogenesis, 2) required for ciliary functions including intraflagellar transport, signal transduction and calcium regulation, 3) localized to the primary cilium or basal body, and 4) mutation in this gene is associated with a known disease referred to as a ciliopathy.
  • FIG. 10 demonstrates the ciliogenesis signature is predictive of cilia-positive staining in NSCLC cell lines.
  • A Human NSCLC cell lines (NCI-H2073, NCI-H2228, NCI-H1975, and NIC-H2030) were grown in vitro and stained for primary cilia using antibodies that recognize acetylated-tubulin (grey-scaled green, ciliary axoneme) and g-tubulin (grey-scaled red, centrosome). Images represents example of NCI-H2228 cells positive for markers of primary cilia (inset). Hoechst dye was used to counterstain nuclei.
  • B Bar graph represents quantification of percentage of cilia-positive NSCLC cells analyzed in A.
  • C Ciliogenesis gene signature score (y-axis) and Hh gene signature score (x-axis) plotted for each of the 47 NSCLC cell lines.
  • gene signature or “gene expression signature” or “gene expression profile” refers to a group of genes in a cell whose combined expression pattern is uniquely characteristic of a biological phenotype or medical condition.
  • treat refers to alleviating or abrogating a disease, e.g., NSCLC, or one or more of the symptoms associated with the disease; or alleviating or eradicating the cause(s) of the disease itself.
  • terapéuticaally effective amount of a compound refers to the amount of a compound (e.g., Hh inhibitor) that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of a disease, e.g., NSCLC, being treated.
  • the term also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.
  • a therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of a disease, e.g., NSCLC.
  • the term encompasses an amount that improves overall therapy, reduces, or avoids symptoms or causes of a disease, e.g., NSCLC, or enhances the therapeutic efficacy of another therapeutic agent.
  • level refers to the amount, accumulation, or rate of a biomarker molecule.
  • a level can be represented, for example, by the amount or the rate of synthesis of a mesenger RNA (mRNA) encoded by a gene, the amount or the rate of synthesis of a polypeptide or protein encoded by a gene, or the amount or the rate of synthesis of a biological molecule accumulated in a cell or biological fluid.
  • mRNA mesenger RNA
  • level refers to an absolute amount of a molecule in a sample or to a relative amount of the molecule, determined under steady-state or non-steady-state conditions.
  • responsiveness refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, e.g., NSCLC, being treated.
  • increased responsiveness when used in reference to a treatment of a cell or a subject refers to an increase in the effectiveness in lessening or decreasing the symptoms of the disease when measured using any methods known in the art.
  • the increase in the effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • an "effective patient response” refers to an increase in the therapeutic benefit to a patient in treating a disease, e.g., NSCLC.
  • the increase is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • An "effective patient tumor response" can be, for example, an about 5%, about 10%, about 25%, about 50%, or about 100% decrease in one or more physical symptoms of the disease or the tumor size.
  • ⁇ refers to an increase in the probability of an event.
  • ⁇ when used in reference to the effectiveness of a patient response to a treatment of a disease, e.g., NSCLC, contemplates an increased probability that the symptoms of the disease will be lessened or decreased.
  • predict generally means to determine or tell in advance.
  • the term “predict” can mean that the likelihood of the outcome of the treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially.
  • determining means determining if an element is present or not.
  • the measurement can be quantitative and/or qualitative determinations. "Assessing the presence of can include determining the amount of something present, as well as determining whether it is present or absent.
  • biological sample refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin, obtained, reached, or collected in vivo or in situ.
  • a biological sample also includes samples from a region of a biological subject containing precancerous or cancer cells or tissues. Such samples can be, but are not limited to, organs, tissues, fractions and cells isolated from a mammal.
  • Exemplary biological samples include, but are not limited to, cell lysate, a cell culture, a cell line, a tissue, oral tissue, gastrointestinal tissue, an organ, an organelle, a biological fluid, a blood sample, a urine sample, a skin sample, and the like.
  • biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, and the like.
  • NSCLC Non-small cell lung cancers
  • Hh oncogenic Hedgehog
  • cilia present or absent profoundly influences tumor response to Hh pathway inhibitors.
  • the presence of cilia normalized to cell cycle
  • such experiments conducted to understand the mechanism of Hh-inhibitor responsiveness suggests a dependence of Vismodegib anti-tumor activity on the presence of cilia.
  • this dependence on cilia is mediated through a post-translational processing of Gli into a transcriptional repressor of tumor growth.
  • biomarkers e.g., presence of primary cilia expression in cancer cells
  • biomarker genes e.g., gene expression profiles
  • a cancer associated with Hh pathway activity e.g., NSCLC
  • methods for stratifying an individual having a cancer associated with Hh pathway activity e.g., NSCLC
  • a cancer associated with Hh pathway activity e.g., NSCLC
  • 7, 8 and/or 9 e.g., ABHD4, ACSS2, ARL14, BLOC1S2, C17orf91, CDKN1A, CLIC3, CLIP3, CYP4F11, DDIT3, DDIT4, GABARAPL1, GCNT2, GDF15, GPX3, GULP1, HKDC1, IDI1, IL1RN, INSIG1, KIAA1370, LCN2, LCP1, LPIN1, MVD, NUPR1, OSGIN1, PANX2, PCSK9, RBCK1, SC4MOL, SCD, SDCBP2, STOX2, TIMP4, TP53INP1, TRIM31, UBC, YPEL5, ARHGAP11A, ASF1B, AURKB, BIRC5, BLM, C15orf42, C16orf59, CASC5, CCNA2, CCNE2, CDC20, CDC6, CENPM, DDN, DLGAP5, DTL, E2F2, EXOl, FAM64A
  • determining the gene expression profile involves quantifying mRNA expression related to the specific biomarker genes.
  • determining the gene expression profile involves quantifying protein expression related to the specific biomarker genes.
  • determining the gene expression profile involves quantifying mRNA expression alnd protein expression related to the specific biomarker genes.
  • the specific gene expression profile for such one or more biomarker genes is for an individual having cancer cells associated with Hh pathway activity and primary cilia expression.
  • the presence of such a specific gene signature indicates that the individual would be responsive to a Hh inhibitor (e.g., a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib) as a treatment of a cancer associated with Hh pathway activity (e.g., NSCLC).
  • a Hh inhibitor e.g., a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib) as a treatment of a cancer associated with Hh pathway activity (e.g., NSCLC).
  • the absence of such a specific gene signature indicates that the individual would not be responsive to a Hh inhibitor (e.g., a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib)) as a treatment of a cancer associated with Hh pathway activity (e.g., NSCLC).
  • a Hh inhibitor e.g., a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib)
  • a cancer associated with Hh pathway activity e.g., NSCLC
  • an individual's therapeutic regimen is optimized, e.g. modifying, discontinuing, or continuing the treatment, based on the presence or absence of such a specific gene expression signature for the one or more biomarker genes selected from Figs. 7, 8 and/or 9.
  • an individual's therapeutic regimen is optimized, e.g.
  • modifying, discontinuing, or continuing the treatment based on the presence or absence of such a specific gene expression signature for the one or more biomarker genes selected from TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS1, PHKHD1, RABL5, HSPBl l, PKDl, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCBl (see, e.g., Example VI).
  • a cancer associated with Hh pathway activity e.g., NSCLC
  • detection of the presence or absence of primary cilia expression in such obtained cancer cells is accomplished with a cilia staining technique (see, e.g., Example VII).
  • the presence of primary cilia expression in such obtained cancer cells indicate that the individual would be responsive to a Hh inhibitor (e.g., a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib)) as a treatment of a cancer associated with Hh pathway activity (e.g., NSCLC).
  • a Hh inhibitor e.g., a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib)
  • an individual's therapeutic regimen is optimized, e.g. modifying, discontinuing, or continuing the treatment, based on the presence or absence of primary cilia expression in such obtained cancer cells.
  • biomarkers e.g., primary cilia expression in cancer cells
  • biomarker gene expression profiles as disclosed herein (e.g., the genes recited in Figs. 7, 8 and/or 9) (e.g., one or more genes selected from TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS1, PHKHD1, RABL5, HSPB11, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1 (see, e.g., Example VI)) for assessing an individual having a cancer associated with Hh pathway activity (e.g., NSCLC) for treatment with a Hh inhibitor (e.g., a SMO inhibitor (e.g., Taladegib (LY2940680),
  • Hh inhibitor e.g., a SMO inhibitor (e.g., Taladegib (LY2940680)
  • the systems comprise the analysis of a biological sample (e.g., a biological sample comprising cancer cells associated with Hh pathway activity; NSCLC cancer cells) by analytical techniques to derive biomarker gene expression profile data and/or analytical measurements.
  • a biological sample e.g., a biological sample comprising cancer cells associated with Hh pathway activity; NSCLC cancer cells
  • biomarker gene expression profile data or analytical measurements are subsequently compiled by software into a dataset, which is then analyzed to determine one or more biomarker indications, such as the presence or absence of a gene expression profile consistent with a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • the results are used to stratify patients prior to or during therapy regiments, to monitor the progress of a therapy regimen, or to optimize a therapy regimen.
  • the results are compiled into a report format for sending to a user.
  • kits and arrays for using biomarkers e.g., primary cilia expression in cancer cells
  • biomarkers e.g., primary cilia expression in cancer cells
  • biomarker gene expression profiles disclosed herein for use with the methods and systems disclosed above.
  • kits disclosed herein comprise one or more reagents for determining the presence or absence of primary cilia expression in obtained cancer cells from an individual (see, e.g., the technique described in Example VII).
  • kits disclosed herein comprise one or more reagents for determining a gene expression profile for the one or more biomarker genes shown in Figs. 7, 8 and/or 9, and a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • kits disclosed herein comprise one or more reagents for determining a gene expression profile for the one or more biomarker genes shown in TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS1, PHKHD1, RABL5, HSPB11, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1, and a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • instructions are provided for comparing the determined gene expression profile with the reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • the present invention provides a nucleic acid hybridization array comprising nucleic acid probes for evaluating if an individual receiving a Hh inhibitor such as a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib) for treatment of a cancer associated with Hh activity (e.g., NSCLC) has developed or is likely to develop resistance to the therapy, comprising nucleic acid probes which hybridize to biomarker genes selected from Figs. 7, 8 and/or 9.
  • a Hh inhibitor such as a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib) for treatment of a cancer associated with Hh activity (e.g., NSCLC)
  • a cancer associated with Hh activity e.g., NSCLC
  • the present invention provides a nucleic acid hybridization array comprising nucleic acid probes for evaluating if an individual receiving a Hh inhibitor such as a SMO inhibitor (e.g., Taladegib (LY2940680), Vismodegib) for treatment of a cancer associated with Hh activity (e.g., NSCLC) has developed or is likely to develop resistance to the therapy, comprising nucleic acid probes which hybridize to biomarker genes selected TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS1, PHKHD1, RABL5, HSPB11, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1.
  • a SMO inhibitor e.g., Taladegib (LY2940680), Vismodegib
  • a cancer associated with Hh activity e.g., NSCLC
  • nucleic acid probes which hybridize to biomarker genes selected TM
  • Cancers associated with Hh pathway activity are a diverse group of cancer that include, but are not limited to, NSCLC, bladder cancer, basal cell carcinoma,
  • medulloblastoma colon cancer
  • breast cancer pancreatic cancer
  • biomarkers or biomarker gene expression profiles for monitoring a patient during treatment of a cancer associated with Hh pathway activity (e.g., NSCLC). Further disclosed herein are methods of using biomarkers or biomarker gene expression profiles for optimizing a treatment regimen.
  • the treatment comprises administration of a Hh inhibitor.
  • a Hh inhibitor is Taladegib (LY2940680).
  • the Hh inhibitor is vismodegib.
  • the Hh inhibitor is selected from Taladegib
  • the Hh inhibitor is selected from Hh ligand inhibitors (e.g., 5E1, robotnikinin), SMO antagonists (e.g., Taladegib (LY2940680), vismodegib, IPI-926, HhAntag), and Gli- processing inhibitors (e.g., HPI-2, HPI-3, HPI-4, arsenic trioxide).
  • Hh ligand inhibitors e.g., 5E1, robotnikinin
  • SMO antagonists e.g., Taladegib (LY2940680
  • vismodegib e.g., IPI-926, HhAntag
  • Gli- processing inhibitors e.g., HPI-2, HPI-3, HPI-4, arsenic trioxide.
  • Mutations or modifications and expression levels of biomarkers are measured by RT-PCR, Qt-PCR, microarray, Northern blot, or other similar technologies. Circulating levels of biomarkers in a blood sample obtained from a candidate subject are measured, for example, by ELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Western blot, multiplexing technologies, or other similar methods. Cell surface expression of biomarkers are measured, for example, by flow cytometry, immunohistochemistry, Western Blot, immunoprecipitation, magnetic bead selection, and quantification of cells expressing either of these cell surface markers.
  • Circulating levels of biomarkers in a blood sample obtained from a candidate subject are measured, for example, by ELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Western blot, multiplexing technologies, or other similar methods.
  • Cell surface expression of biomarkers are measured, for example, by flow cytometry, immunohistochemistry, Western Blo
  • determining the presence, modifications, or expression of the biomarker of interest at the protein or nucleotide level are accomplished using any detection method known to those of skill in the art.
  • determining the modification(s) is intended to determine a mutation within the biomarker gene or a biomarker protein.
  • biomarker refers to in some cases the protein of interest. In some cases, “biomarker” refers to the gene of interest. In some cases, the terms “biomarker” and “biomarker gene” are used interchangeably.
  • detecting expression or “detecting the level of is intended determining the expression level or presence of a biomarker protein or gene in the biological sample. Thus, “detecting expression” encompasses instances where a biomarker is determined not to be expressed, not to be detectably expressed, expressed at a low level, expressed at a normal level, or overexpressed.
  • the modifications, expression, or presence of these various biomarkers and any clinically useful prognostic markers in a biological sample are detected at the protein or nucleic acid level, using, for example, immunohistochemistry techniques or nucleic acid-based techniques such as in situ hybridization and RT-PCR.
  • the modifications, expression, or presence of one or more biomarkers is carried out by a means for nucleic acid amplification, a means for nucleic acid sequencing, a means utilizing a nucleic acid microarray (DNA and RNA), or a means for in situ hybridization using specifically labeled probes.
  • the determining the modification, expression, or presence of one or more biomarkers is carried out through gel electrophoresis. In one embodiment, the determination is carried out through transfer to a membrane and hybridization with a specific probe.
  • the determining the modification, expression, or presence of one or more biomarkers carried out by a diagnostic imaging technique.
  • the determining the modification, expression, or presence of one or more biomarkers carried out by a detectable solid substrate is paramagnetic nanoparticles functionalized with antibodies.
  • Methods for detecting the modification and expression of the biomarkers described herein, within the test and control biological samples comprise any methods that determine the quantity or the presence of these markers either at the nucleic acid or protein level. Such methods are well known in the art and include but are not limited to western blots, northern blots, ELISA, immunoprecipitation, immunofluorescence, flow cytometry,
  • expression of a biomarker is detected on a protein level using, for example, antibodies that are directed against specific biomarker proteins. These antibodies are used in various methods such as Western blot, ELISA, multiplexing technologies, immunoprecipitation, or immunohistochemistry techniques.
  • detection of biomarkers is accomplished by ELISA.
  • detection of biomarkers is accomplished by electrochemiluminescence (ECL).
  • the modification, expression, or presence of one or more of the biomarkers described herein are determined at the nucleic acid level.
  • Nucleic acid-based techniques for assessing expression are well known in the art and include, for example, determining the level of biomarker mRNA in a biological sample. Many expression detection methods use isolated RNA. Any RNA isolation technique that does not select against the isolation of mRNA is utilized for the purification of RNA (see, e.g., Ausubel et al, ed. (1987- 1999) Current Protocols in Molecular Biology (John Wiley & Sons, New York).
  • tissue samples are readily processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process disclosed in U. S. Pat. No. 4,843,155.
  • nucleic acid probe refers to any molecule that is capable of selectively binding to a specifically intended target nucleic acid molecule, for example, a nucleotide transcript. Probes are synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes are specifically designed to be labeled, for example, with a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or other labels or tags that are discussed above or that are known in the art. Examples of molecules that are utilized as probes include, but are not limited to, RNA and DNA.
  • isolated mRNA are used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays.
  • One method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that hybridize to the mRNA encoded by the gene being detected.
  • the nucleic acid probe comprises of, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a biomarker, biomarker described herein above.
  • Hybridization of an mRNA with the probe indicates that the biomarker or other target protein of interest is being expressed.
  • the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in a gene chip array.
  • a skilled artisan readily adapts known mRNA detection methods for use in detecting the level of mRNA encoding the biomarkers or other proteins of interest.
  • An alternative method for determining the level of an mRNA of interest in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (see, for example, U. S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88: 189 193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci.
  • biomarker expression is assessed by quantitative fluorogenic RT- PCR (i.e., the TaqManO System).
  • RNA of interest Modifications or expression levels of an RNA of interest are monitored using a membrane blot (such as used in hybridization analysis such as Northern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U. S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677, 195 and 5,445,934, which are incorporated herein by reference.
  • the detection of expression also comprises using nucleic acid probes in solution.
  • microarrays are used to determine expression or presence of one or more biomarkers. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible partem of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, U. S. Pat. Nos.
  • High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
  • Exemplary microarray chips include FoundationOne and FoundationOne Heme from Foundation Medicine, Inc; GeneChip® Human Genome U133 Plus 2.0 array from Affymetrix; and Human
  • an array is fabricated on a surface of virtually any shape or even a multiplicity of surfaces.
  • an array is a planar array surface.
  • arrays include peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U. S. Pat. Nos. 5,770,358, 5,789, 162, 5,708,153, 6,040, 193 and 5,800,992, each of which is hereby incorporated in its entirety for all purposes.
  • arrays are packaged in such a manner as to allow for diagnostics or other manipulation of an all- inclusive device.
  • any means for specifically identifying and quantifying a biomarker in the biological sample of a candidate subject is contemplated.
  • expression level of a biomarker protein of interest in a biological sample is detected by means of a binding protein capable of interacting specifically with that biomarker protein or a biologically active variant thereof.
  • labeled antibodies, binding portions thereof, or other binding partners are used.
  • label when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody.
  • the label is detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, catalyzes chemical alteration of a substrate compound or composition that is detectable.
  • the antibodies for detection of a biomarker protein are either monoclonal or polyclonal in origin, or are synthetically or recombinantly produced.
  • the amount of complexed protein for example, the amount of biomarker protein associated with the binding protein, for example, an antibody that specifically binds to the biomarker protein, is determined using standard protein detection methodologies known to those of skill in the art.
  • a detailed review of immunological assay design, theory and protocols are found in numerous texts in the art (see, for example, Ausubel et al, eds. (1995) Current Protocols in Molecular Biology) (Greene Publishing and Wiley-Interscience, NY)); Coligan et al, eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, N.Y.).
  • the choice of marker used to label the antibodies will vary depending upon the application. However, the choice of the marker is readily determinable to one skilled in the art.
  • These labeled antibodies are used in immunoassays as well as in histological applications to detect the presence of any biomarker or protein of interest.
  • the labeled antibodies are either polyclonal or monoclonal.
  • the antibodies for use in detecting a protein of interest are labeled with a radioactive atom, an enzyme, a chromophoric or fluorescent moiety, or a colorimetric tag as described elsewhere herein.
  • the choice of tagging label also will depend on the detection limitations desired. Enzyme assays (ELISAs) typically allow detection of a colored product formed by interaction of the enzyme-tagged complex with an enzyme substrate.
  • Radionuclides that serve as detectable labels include, for example, 1 -131, 1-123, 1-125, Y-90, Re-188, Re-186, At-211 , Cu-67, Bi-212, and Pd-109.
  • enzymes that serve as detectable labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose-6-phosphate dehydrogenase.
  • Chromophoric moieties include, but are not limited to, fluorescein and rhodamine.
  • the antibodies are conjugated to these labels by methods known in the art.
  • enzymes and chromophoric molecules are conjugated to the antibodies by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like.
  • conjugation occurs through a ligand-receptor pair.
  • suitable ligand-receptor pairs are biotin- avidin or biotin-streptavidin, and antibody-antigen.
  • expression or presence of one or more biomarkers or other proteins of interest within a biological sample is determined by radioimmunoassays or enzyme-linked immunoassays (ELISAs), competitive binding enzyme-linked immunoassays, dot blot (see, for example, Promega Protocols and Applications Guide, Promega Corporation (1991), Western blot (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Vol. 3, Chapter 18 (Cold Spring Harbor Laboratory Press, Plainview, N.Y.), chromatography such as high performance liquid chromatography (HPLC), or other assays known in the art.
  • the detection assays involve steps such as, but not limited to, immunoblotting, immunodiffusion, Immunoelectrophoresis, or immunoprecipitation.
  • the sample for use in the methods is obtained from cells of a cancer cell line (e.g., a cancer associated with Hh pathway activity).
  • the sample is obtained from cells of NSCLC, basal cell carcinoma, pancreatic cancer, medulloblastoma, colon cancer, breast cancer, and/or bladder cancer.
  • the sample for use in the methods is from any tissue or fluid from a patient.
  • Samples include, but are not limited, to whole blood, dissociated bone marrow, bone marrow aspirate, pleural fluid, peritoneal fluid, central spinal fluid, abdominal fluid, pancreatic fluid, cerebrospinal fluid, brain fluid, ascites, pericardial fluid, urine, saliva, bronchial lavage, sweat, tears, ear flow, sputum, hydrocele fluid, semen, vaginal flow, milk, amniotic fluid, and secretions of respiratory, intestinal or genitourinary tract.
  • the sample is a blood serum sample.
  • the sample is from a fluid or tissue that is part of, or associated with, the lymphatic system or circulatory system.
  • the sample is a blood sample that is a venous, arterial, peripheral, tissue, cord blood sample.
  • the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the sample contains one or more circulating tumor cells (CTCs).
  • CTCs circulating tumor cells
  • DTC disseminated tumor cells
  • the samples are obtained from the individual by any suitable means of obtaining the sample using well-known and routine clinical methods.
  • Procedures for obtaining fluid samples from an individual are well known. For example, procedures for drawing and processing whole blood and lymph are well-known and can be employed to obtain a sample for use in the methods provided.
  • an anti-coagulation agent e.g., EDTA, or citrate and heparin or CPD (citrate, phosphate, dextrose) or comparable substances
  • the blood sample is collected in a collection tube that contains an amount of EDTA to prevent coagulation of the blood sample.
  • the collection of a sample from the individual is performed at regular intervals, such as, for example, one day, two days, three days, four days, five days, six days, one week, two weeks, weeks, four weeks, one month, two months, three months, four months, five months, six months, one year, daily, weekly, bimonthly, quarterly, biyearly or yearly.
  • the collection of a sample is performed at a predetermined time or at regular intervals relative to treatment with a Hh inhibitor (e.g., SMO inhibitor).
  • a Hh inhibitor e.g., SMO inhibitor
  • a sample is collected from a patient at a predetermined time or at regular intervals prior to, during, or following treatment or between successive treatments with a Hh inhibitor.
  • a sample is obtained from a patient prior to administration of a Hh inhibitor, and then again at regular intervals after treatment with the Hh inhibitor has been effected.
  • the patient is administered a Hh inhibitor and one or more additional therapeutic agents.
  • a Hh inhibitor is administered in combination with an additional therapeutic agent for the treatment of a cancer associated with Hh pathway activity.
  • the Hh inhibitor includes Hh ligand inhibitors (e.g., 5E1, robotnikinin), SMO antagonists (e.g., vismodegib, IPI-926, HhAntag), and Gli-processing inhibitors (e.g., HPI-2, HPI-3, HPI-4, arsenic trioxide).
  • an Hh inhibitor is administered in combination with an additional therapeutic agent for the treatment of a cancer associated with Hh pathway activity (e.g., NSCLC).
  • the additional therapeutic agent including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g. , surgical intervention, and/or radiotherapies).
  • the additional therapeutic agent(s) is an anticancer agent.
  • anticancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis;
  • polynucleotides e.g. , anti-sense, ribozymes, siRNA
  • polypeptides e.g. , enzymes and antibodies
  • biological mimetics alkaloids; alkylating agents; antitumor antibiotics;
  • antimetabolites include hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides;
  • biological response modifiers e.g., interferons (e.g. , IFN-a) and interleukins (e.g. , IL-2)
  • adoptive immunotherapy agents hematopoietic growth factors; agents that induce tumor cell differentiation (e.g. , all-trans-retinoic acid); gene therapy reagents (e.g. , antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors; proteosome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like.
  • chemotherapeutic compounds and anticancer therapies suitable for coadministration with Hh inhibitors are known to those skilled in the art.
  • anticancer agents comprise agents that induce or stimulate apoptosis.
  • Agents that induce apoptosis include, but are not limited to, radiation (e.g. , X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-Rl or TRAIL-R2); kinase inhibitors (e.g.
  • epidermal growth factor receptor (EGFR) kinase inhibitor vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet-derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g. , HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); anti- androgens (e.g.
  • cyclooxygenase 2 (COX-2) inhibitors e.g. , celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)
  • anti -inflammatory drugs e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan
  • C AMPTOS AR CPT- 11 , fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP- 16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.
  • compositions and methods of the present invention provide a Hh inhibitor and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds).
  • Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and
  • methylmelamines e.g. , hexamethylmelamine and thiotepa
  • alkyl sulfonates e.g., busulfan
  • nitrosoureas e.g. , carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)
  • triazenes e.g., dacarbazine (DTIC;
  • antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g., methotrexate (amethopterin)); 2) pyrimidine analogs (e.g. , fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g. , mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin)).
  • folic acid analogs e.g., methotrexate (amethopterin)
  • pyrimidine analogs e.g. , fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cy
  • chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin
  • vinca alkaloids e.g., vinblastine (VLB), vincristine
  • epipodophyllotoxins e.g., etoposide and teniposide
  • antibiotics e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin
  • мmycin C (mitomycin C)); 4) enzymes (e.g. , L-asparaginase); 5) biological response modifiers (e.g., interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted ureas (e.g. , hydroxyurea); 9) methylhydrazine derivatives (e.g. , procarbazine (N-methylhydrazine;
  • enzymes e.g. , L-asparaginase
  • biological response modifiers e.g., interferon-alfa
  • platinum coordinating complexes e.g., cisplatin (cis-DDP) and carboplatin
  • anthracenediones e.g., mitoxantrone
  • substituted ureas
  • adrenocortical suppressants e.g., mitotane ( ⁇ , ⁇ '-DDD) and aminoglutethimide
  • 11 adrenocorticosteroids
  • 12 progestins (e.g. , hydroxy progesterone caproate, medroxyprogesterone acetate, and megestrol acetate)
  • 13 estrogens (e.g. , diethylstilbestrol and ethinyl estradiol)
  • antiestrogens e.g. , tamoxifen
  • 15) androgens e.g. , testosterone propionate and fluoxymesterone
  • 16 antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g. , leuprolide).
  • any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention.
  • the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States.
  • International counterpart agencies to the U.S.F.D.A. maintain similar formularies.
  • Table 1 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents. Table 1
  • Cisplatin Platinol Bristol-Myers Squibb (PtCI 2 H e N 2 )
  • Epoetin alfa Epogen Amgen, Inc (recombinant peptide)
  • Methotrexate Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid)
  • Tositumomab I 131 Tositumomab Bexxar Corixa Corp., Seattle, (recombinant murine immunotherapeutic monoclonal lgG 2a WA
  • Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-0- tetradecanoylphorbol- 13 -acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI- PEG 20, AE-941, AG-013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combreta
  • VNP40101M volociximab
  • vorinostat VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.
  • anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al , 2002.
  • the present invention provides methods for administering a Hh inhibitor with radiation therapy.
  • the invention is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal.
  • the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof.
  • the radiation is delivered to the animal using a linear accelerator.
  • the radiation is delivered using a gamma knife.
  • the source of radiation can be external or internal to the animal.
  • External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals.
  • Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g. , using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive.
  • Types of internal radiation therapy include, but are not limited to, brachy therapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.
  • the animal may optionally receive radiosensitizers (e.g. , metronidazole,
  • misonidazole intra-arterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5- substituted-4-nitroimidazoles, 2H-isoindolediones, [ [(2 -bromoethyl)-amino] methyl] -nitro- lH-imidazole-l-ethanol, nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine- containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5- thiotretrazole derivative, 3-nitro-l,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea,
  • Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.
  • Radiotherapy any type of radiation can be administered to an animal, so long as the dose of radiation is tolerated by the animal without unacceptable negative side-effects.
  • Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g. , high linear energy radiation).
  • Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e. , gain or loss of electrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference in its entirety).
  • the effects of radiation can be at least partially controlled by the clinician.
  • the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.
  • the total dose of radiation administered to an animal is about .01 Gray (Gy) to about 100 Gy.
  • about 10 Gy to about 65 Gy e.g. , about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy
  • a complete dose of radiation can be administered over the course of one day, the total dose is ideally fractionated and
  • radiotherapy is administered over the course of at least about 3 days, e.g. , at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks).
  • a daily dose of radiation will comprise approximately 1-5 Gy (e.g. , about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2 Gy (e.g. , 1.5-2 Gy).
  • the daily dose of radiation should be sufficient to induce destruction of the targeted cells.
  • radiation is not administered every day, thereby allowing the animal to rest and the effects of the therapy to be realized.
  • radiation desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week.
  • radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the animal's responsiveness and any potential side effects.
  • Radiation therapy can be initiated at any time in the therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of the therapeutic period.
  • radiation is administered in weeks 1-6 or in weeks 2-6 of a therapeutic period comprising 6 weeks for treating, for instance, a solid tumor.
  • radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks.
  • Antimicrobial therapeutic agents may also be used as therapeutic agents in the present invention. Any agent that can kill, inhibit, or otherwise attenuate the function of microbial organisms may be used, as well as any agent contemplated to have such activities.
  • Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g., defensins), antisense nucleic acids, membrane disruptive agents and the like, used alone or in combination. Indeed, any type of antibiotic may be used including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like.
  • systems of assessing an individual having a cancer associated with Hh pathway activity comprising: (a) a digital processing device comprising an operating system configured to perform executable instructions, and an electronic memory; (b) a dataset stored in the electronic memory, wherein the dataset comprises data for one or more biomarker gene expression profiles in a sample; and (c) a computer program including instructions executable by the digital processing device to create an application comprising: (i) a first software module configured to analyze the dataset for similarity with a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression; and (ii) a second software module to assign the individual as a candidate for treatment with a Hh inhibitor if there is a similarity between the determined gene expression profile and a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.
  • a digital processing device comprising an operating system configured to perform executable instructions, and an electronic memory
  • a dataset stored in the electronic memory wherein the dataset comprises data for one or more biomarker gene expression profiles in
  • the systems and methods described herein include a digital processing device, or use of the same.
  • the digital processing device includes one or more hardware central processing units (CPU) that carry out the device's functions.
  • the digital processing device further comprises an operating system configured to perform executable instructions.
  • the digital processing device is optionally connected to a computer network.
  • the digital processing device is optionally connected to the Intemet such that it accesses the World Wide Web.
  • the digital processing device is optionally connected to a cloud computing infrastructure.
  • the digital processing device is optionally connected to an intranet.
  • the digital processing device is optionally connected to a data storage device.
  • suitable digital processing devices include, by way of non-limiting examples, server computers, desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • server computers desktop computers, laptop computers, notebook computers, sub-notebook computers, netbook computers, netpad computers, set-top computers, media streaming devices, handheld computers, Internet appliances, mobile smartphones, tablet computers, personal digital assistants, video game consoles, and vehicles.
  • smartphones are suitable for use in the system described herein.
  • Suitable tablet computers include those with booklet, slate, and convertible configurations, known to those of skill in the art.
  • Hh-targeted inhibitors predicted to be cilia-dependent include, but are not limited to, Hh-ligand antagonists (e.g. 5E1 or robotnikinin), Smo antagonists (e.g. Vismodegib, IPI- 926, HhAntag), and Gli-processing inhibitors (e.g. HPI-2,3) (Fig. 2). These inhibitors all involve inhibiting components of the Hh pathway that require cilia (see, e.g., Hassounah, N.B., et al., 2012 Clin Cancer Res 18, 2429-2435). Therefore, it is predicted that these inhibitors will be effective only if the tumor cells activate the Hh pathway via a cilia- dependent mechanism.
  • Hh-ligand antagonists e.g. 5E1 or robotnikinin
  • Smo antagonists e.g. Vismodegib, IPI- 926, HhAntag
  • Gli-processing inhibitors e.g. HPI-2,3
  • Vismodegib is a Smo inhibitor and was recently shown to cause rapid tumor regression in patients with advanced basal cell carcinoma (BCC) (see, e.g., Von Hoff, D.D., et al, 2009 N Engl J Med 361, 1164-1172). Vismodegib was the first Hh inhibitor to be approved by the FDA for treatment of BCC. However, Vismodegib is not efficacious in all BCC patients and some patients that initially responded to treatment have been documented to relapse (see, e.g., Chang, A.L., et al, (2014) J Am Acad Dermatol 70, 60-69; Von Hoff, D.D., et al, 2009 N Engl J Med 361, 1164-1172).
  • the percentage of ciliated cancer cells associated with response to the Smo inhibitor Vismodegib was analyzed.
  • Primary BCC tumor samples from patients on a clinical trial with Vismodegib (Von Hoff, D.D., et al, 2009 N Engl J Med 361, 1164-1172) were stained. The patient samples were taken prior to treatment with Vismodegib and were stained for markers of primary cilia and then normalized to cell cycle (%Ki67 positive cancer cells) for each patient to obtain a cilia score. It was found that BCC patients with best response (complete response) to Vismodegib have the highest cilia score (median: 86%) compared to patients with partial response (median: 66%), stable disease
  • High Hh activity has been associated with poor overall survival and decreased relapse- free survival in many cancers including BCC, bladder cancer, pancreatic cancer, medulloblastoma and NSCLC (see, e.g., Raz G, et al, 76 Lung Cancer 2012, pages 191-196; Chen, M., et al, 2010 Cancer Prev Res (Phila) 3, 1235-1245; Dai, J., et al., 2011 Pancreas 40, 233-236; Al-Halabi, H., 2011 Acta Neuropathol 121, 229-239). Therefore, such results indicate a significant impact by making important advances in the therapy of the many cancer types with upregulated Hh signaling.
  • Hh signaling pathway is active in -50% of early -stage NSCLC patient samples (see, e.g., Raz G, et al, 76 Lung Cancer 2012, pages 191-196).
  • Ptch a Hh responsive gene indicating increased Hh signaling
  • overall survival and decreased relapse-free survival Such data suggest that these patients would benefit from treatment with a Hh inhibitor.
  • the data on primary cilia highlight the importance of determining if NSCLC activate the Hh pathway via a cilia-dependent mechanism. Determining the general prevalence of NSCLC patients whose tumors are positive or negative for cilia will aid in the design and selection of patients for Hh-targeted therapies. Experiments conducted during the course of developing embodiments for the present invention strongly suggests that this strategy will expand the field of use of the Hh inhibitor Vismodegib.
  • Hh activity is characterized by increased transcription and expression of Glil and Ptchl .
  • Experiments conducted during the course of developing embodiments for the present invention demonstrates that of NSCLC cases with >25% staining intensity (based on general intensity scoring) of Glil and/or Ptchl there was a significant correlation with Smo
  • a NSCLC cell line (A549 cells) was identified that is responsive to the cilia-dependent Smo inhibitor Vismodegib (see, e.g., Shi, S., et al, 2012 J Biol Chem 287, 7845-7858).
  • A549 cells were obtained and utilized to demonstrate that these cells express primary cilia (Fig. 5B).
  • Such data supports the hypothesis that Hh signaling, in a subset of Hh-active NSCLC patients, is activated via a cilia-dependent mechanism.
  • Tissue Clinically annotated NSCLC samples for expression of primary cilia will be examined. Such exmperiments will also correlate this with expression of Glil, Ptchl, Smo and Shh.
  • TMA tissue microarrays
  • the TMAs have duplicate 1mm cores representing a total of 245 NSCLC cases.
  • stage I disease had stage I disease and the rest are stage II disease.
  • stage II disease 93.6% of the NSCLCs are from current or former smokers. These patients were diagnosed between 2001 and 2007 and they are fully annotated for age at diagnosis, gender, histologic subtype, stage, smoking history, relapse- free survival and overall survival.
  • the second serial sections will be stained with antibodies to detect cilia (anti-acetylated-tubulin antibody to recognize the ciliary axoneme and anti-gamma (y)-tubulin antibody to recognize the centrosome) and to co-stain for a marker of activated Hh signaling (anti-Ptchl antibody or anti-Glil antibody).
  • Ptchl was chosen as the third marker because 48.5% of the 245 NSCLC cases were shown to be Ptchl positive and these had a significant correlation with Glil, Smo and Shh.
  • These three primary antibodies are all anti-mouse antibodies but all have different isotypes, which allows us to utilize three different fluorescently conjugated secondary antibodies (Alexa-488, Alexa-564 and Alexa-633) to distinguish between primary antibodies.
  • All tissue cores will be imaged using a Leica Confocal Microscope located at the University of Arizona Cancer Center. Such experiments will image with a 60X objective and obtain three fields/core for each patient to obtain maximum coverage. See Fig. 1 for example of fluorescent cilia staining in paraffin tissue sections from the
  • NSCLC cohort The third serial section will be stained with an antibody to recognize Ki67 as a marker of proliferation.
  • Ki67 an antibody to recognize Ki67 as a marker of proliferation.
  • Such experiments will use standard immunohistological staining of each TMA for Ki67 as done previously (see, e.g., Hassounah, N.B., et al, 2013 PLoS ONE 8, e68521).
  • the Ptchl -positive and Ptchl -negative cancer cells with cilia will be scored by manually counting cilia per nuclei and calculating the percentage of cancer cells that are both cilia-positive and Ptch- positive. Note that a cell will only be considered cilia positive if both acetylated and ⁇ -tubulin signal are detected together.
  • the serial adjacent slides stained for Ki67 will be digitally scanned and analyzed using
  • Definiens Software This software allows for unbiased, automated scoring of the fraction of Ki67 positive nuclei in regions of interest. Cilia are found on cells in GO or early Gl of the cell cycle. Therefore, low percent cilia score could be due to a high percentage of proliferative cells. In order to interpret the percentage of cilia such experiments will add the percent cilia of cancer cells to the percent Ki67 for each imaged region to obtain a normalized 'cilia score'. Thus, a low 'cilia score' will indicate loss of cilia expression for reasons unrelated to increased cell proliferation.
  • Cilia scores will be grouped into four categories (>25%, 25-50%, 51-75% and 76- 100%). Such experiments will correlate the four cilia score categories directly with Ptchl positivity. Such experiments will also utilize previous data generated on these TMAs to correlate cilia score categories with Glil, Smo and Shh staining intensity (see, e.g., Raz G, et al., 76 Lung Cancer 2012, pages 191-196). The 4 cilia score categories will also be correlated with overall survival and relapse-free survival to determine if cilia status influences the aggressiveness of the disease and is useful as a novel prognostic marker.
  • Hh-positive NSCLC has a high percentage of ciliated cancer cells further supporting the hypothesis.
  • pathologic subtype adenocarcinoma, squamous cell carcinoma and other
  • age at diagnosis gender
  • smoking history disease stage
  • relapse-free survival overall survival.
  • High Hh activity has been associated with poor overall survival and decreased relapse-free survival in bladder cancer, pancreatic adenocarcinoma and medulloblastoma (see, e.g.,
  • Hh-active NSCLC samples have a high percentage of ciliated cancer cells will provide justification to move forward with a clinical trial using the FDA approved Hh-targeted drug Vismodegib (cilia-dependent) and preliminary data supports this expectation. Indeed, preliminary data suggests that -36% of NLCLC cases are cilia- positive. Because the preliminary data indicates that not all NLCLC cases are cilia-positive it is predicted that the results will demonstrate absence of cilia in a subset of NSCLC cases.
  • Hh signaling promotes proliferation and tumorigenesis in vitro and in vivo (see, e.g., Schidlow, D.V. (1994) Ann Allergy 73, 457-468; quiz 468-470).
  • small molecule inhibitors Vismodegib, HhAntag, GANT61
  • shRNAs targeting Glil or Smo were able to decrease cell viability and cell migration of NSCLC in vitro and reduce tumor growth of NSCLC models in vivo.
  • Experiments conducted for the present invention demonstrated that, within a NSCLC cohort, high Hh activity correlated with decreased overall survival and decreased relapse-free survival.
  • Hh signaling can be activated in cancer cells in a cilia-dependent and cilia-independent manner.
  • NSCLC cell lines A NSCLC cell line (A549) that has high Hh signaling has been identified.
  • Hh inhibitor Vismodegib see, e.g., Maitah, M.Y., et al, 2011 PLoS ONE 6, el6068. Consistent with this hypothesis, such experiments determined that A549 cells are cilia-positive (Fig. 5B). Experiments will use shRNAs that knock down genes required for ciliogenesis to determine if Hh activation is dependent on cilia in A549 cells and if Hh inhibitors require cilia for drug sensitivity in vitro.
  • shRNAs have been designed that when individually knocked down result in loss of ciliogenesis: CCDC41, IFT88 and CEP164. Use of shRNAs to more than one ciliogenesis gene will allow determine if that drug efficacy is related to cilia in general and not to a non-ciliary function of these specific genes. Scramble shRNA will also be used as a control. Control (cilia-positive) and experimental (cilia-negative) A549 cells will be analyzed for Hh activity and cell viability following treatment with Vismodegib. Such experiments will also determine the IC50/LD50 of Vismodegib in the control and experimental groups to determine if loss of cilia affects sensitivity.
  • qPCR will be performed to determine if control and experimental A549 cells treated with drug have expression of Hh target genes including Glil, Gli2, and Ptchl .
  • Cell viability will be assessed using a standard MTT assay.
  • Such experiments also propose to screen a panel of NSCLC cell lines for expression of primary cilia that are known to have high Hh signaling (including: NCI-H520, NCI-H226, SK-MES-1) (see, e.g., Huang, L., et al., 2014 Clin Cancer Res 20, 1566-1575).
  • Such experiments will then use the same shRNAs in the cilia-positive cell lines to assess the role of cilia in mediating Hh inhibitor efficacy more globally in NSCLC.
  • LAC adenocarcinoma
  • chemo-radiation naive human patient LAC tumors that have been found to harbor either high or low scores for primary cilia expression (Fig. 5).
  • Fig. 5 Maintained via in vivo serial propagation, these LAC explants maintain both the genetic and histological characteristics of the original patient tumor.
  • Such experiments will use LAC explants in the conventional heterotopic (flank) position, which allows assessment of therapeutic response via measurement with calipers.
  • mice will undergo in vivo image analysis using a Pearl Impluse imager and a fluorescent dye, which preferentially marks tumor cells, to qualitatively assess tumor burden.
  • H&E staining H&E staining
  • GUI Hh activity
  • Ki-67 staining proliferation
  • apoptosis cleaved caspase 3 staining
  • Tumor response in vivo It is expected that Vismodegib will reduce tumor growth more effectively in the LAC PDX with a high cilia score as compared to the LAC PDX with low cilia score.
  • the LAC PDX with a high cilia score (93%) is a similar score to that resulted in complete response in BCC.
  • the LAC PDX with a low cilia score (54%) is similar to a BCC score that resulted in minimal response (progressive or stable disease).
  • Fig. 7 shows a list of genes upregulated when the cancer cells are treated with GANT61 (an inhibitor of GLI1 and GLI2, both of which are transcriptional regulators of Hh signaling). Therefore, these genes would have downregulated expression when Hh is ON.
  • Fig. 8 shows a list of genes downregulated when the cancer cells are treated with GANT61 Inhibitor (an inhibitor of GLI1 and GLI2, both of which are transcriptional regulators of Hh signaling). Therefore, these genes would have upregulated expression when Hh is ON.
  • GANT61 Inhibitor an inhibitor of GLI1 and GLI2, both of which are transcriptional regulators of Hh signaling. Therefore, these genes would have upregulated expression when Hh is ON.
  • Fig. 9 shows a list of genes that are are associated with primary cilia in one or more of the following ways: 1) required for ciliogenesis, 2) required for ciliary functions including intraflagellar transport, signal transduction and calcium regulation, 3) localized to the primary cilium or basal body, and 4) mutation in this gene is associated with a known disease referred to as a ciliopathy.
  • This example demonstrates gene expression signatures representing Hh pathway activity and ciliogenesis.
  • Hh-targeted drug efficacy in NSCLC was utilized.
  • a bioinformatics approach was utilized to identify cancer patients that have expression of genes associated with the Hh pathway and with ciliogenesis.
  • Hh-target gene expression signature score was developed that when "high” represents activation of signaling by the Hh pathway.
  • the gene signature is based on the 40 gene expression values that are known to go up (Fig.8) or the 52 gene expression values that are known to go down (Fig. 7) when cancer cells were treated with the Hh inhibitor GANT61
  • ⁇ Hh pathway activity score (for each patient) (median of the x GANT DOWN gene expression values) - (median of the x GANT UP gene expression values).
  • a ciliogenesis gene expression signature was derived that can be used to predict frequency of primary cilia in cancers (see list of ciliogenesis genes).
  • a published NSCLC microarray dataset was analyzed for all of the ciliogenesis genes listed in Fig. 9 (see, e.g., Hou J, Aerts J, et al, PLoS ONE. 2010;5(4):el0312).
  • the top 18 ciliogenesis genes expressed in NSCLC as compared to normal control lung samples were identified.
  • This subset of ciliogenesis genes includes TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS 1, PHKHD1, RABL5, HSPB11, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1.
  • a ciliogenesis gene expression score was calculated for each patient using the following formula:
  • Ciliogenesis score (for each patient) (median of the 18 ciliogenesis gene expression values).
  • NSCLC NSCLC
  • a scatter-plot was utilized to determine if there is a correlation between ciliogenesis and Hh pathway activity.
  • Hh pathway activity score was plotted on the x axis and the ciliogenesis score was plotted on the y axis to examine the relationship between the two expression scores.
  • This example shows an experimental method for cilia staining and scoring (see, e.g., Raz G, et al., 76 Lung Cancer 2012, pages 191-196).
  • Paraffin-embedded tissue slides were deparaffinized in a dry incubator at 65°C for 15 minutes and hydrated by washing with Xylene (2 x 10 min), 100% Isopropanol (2 x 10 min), 70% Isopropanol (2 x 10 min), 50% Isopropanol (2 x 10 min), and ultrapure water (2 x 10 min). All washes were at room temperature. Antigen retrieval with a ImM EDTA unmasking solution was performed using a 2100 Retriever (Electron Microscopy Sciences) according to manufacturer's instructions.
  • Tissue slides were placed in Shandon Coverplates (Thermo Scientific, Cat# 72-110-017,) and then into Sequenza Slide Racks (Thermo Scientific, Cat# NC0263065). Tissue slides were blocked with ChemMate Antibody Dilution Buffer (Ventana Medical Systems, Inc., Cat#ADB250) with goat serum (5%) (Invitrogen Corporation, Cat# 16210-064) for 45 minutes at room temperature. Primary and secondary antibodies were diluted in the
  • Primary antibodies were used against acetylated tubulin (mouse monocloncal IgG2B, Sigma, Cat# T7451, clone 6-11B-1), ⁇ -tubulin (mouse monoclonal Igd, Sigma, Cat# T5326, clone GTU-88), Arl 13b (1 : 300, mouse monoclonal igG 2a , UC, Davis/NIH NeuroMab Facility, clone N2.95B/66), and incubated on the tissue overnight at 4°C. The slides were then washed with PBS for 10 minutes (3 x 10 min).
  • the secondary antibodies used were tetramethylrhodamine isothiocyanate (TRITC)- labeled goat anti-mouse-IgG 2 B (Southern Biotech, Cat# 1090-03), Alexa 633-labeled goat anti-mouse-IgGi (Invitrogen, Cat# A21126), and Alexa 546-labeled goat anti-mouse-IgG2 a (Invitrogen, Cat#A21133). Secondary antibodies were incubated on the tissue for 45 minutes at room temperature. Slides were washed with PBS for 10 minutes (3 x 10 min).
  • TRITC tetramethylrhodamine isothiocyanate
  • Hoechst 33342 (Cat#H3570, Invitrogen) was used as a counterstain at 1 : 1000 and incubated on slides for 10 minutes, followed by washing with PBS for 5 minutes (2 x 10 min). Slides were mounted with 1.5 coverslips (0.16-0.19 mm thickness) (Fisher Scientific, Cat# 12-544B) using Prolong Gold Antifade mounting media (Cat# P36934, Invitrogen).
  • Z stacks were then acquired with the violet-laser diode at 405 nm to detect Hoechst staining at a total thickness of 2 ⁇ 0.5 ⁇ , with a Z-step taken every 1 ⁇ .
  • Cilia were then imaged within these areas of interest using a 63x objective (1.4 NA PL Apo) with the helium neon lasers (543 nm and 633 nm), the argon laser (488 nm), and the violet-laser diode (405 nm) was used to detect Hoechst staining.
  • Z-stacks were acquired at a total thickness of 5.0 ⁇ 0.5 ⁇ , with a Z-step taken every 0.34 ⁇ (image resolution 2048x2048 pixels). Such experiments acquired a range of 3-6 images per location using the 63x objective per tissue type per patient and this varied depending on the size of the location. Z images were processed post-acquisition to maximum projections using the Leica LAS AF software for image analysis.
  • Cilia frequency and cilia lengths were obtained for each cell type using the Leica LAS AF software. Cilia were only scored when both ciliary axoneme and centrosome were visible together. For each cell nuclei were counted using the count tool, and cilia lengths were measured using the scale bar tool. The number of cilia per cell type was divided by total nuclei per cell type to obtain a percentage of ciliated cells.
  • Hematoxylin 1 (Thermo Scientific, Cat# 7221) was used to counterstain the tissue slides. Hematoxylin 1 was diluted 1 :3 and incubated on tissue slides for 15 seconds and then rinsed in tap water until water ran clear. Faramount Aqueous Mounting Media (Dako, Cat# S3025) was used for mounting slides using 1.5 coverslips (0.16-0.19 mm thickness) (Fisher Scientific, Cat# 12-544B).
  • Intraclass correlation A statistical test that is used to measure the consistency and absolute agreement of measurements made by different observers (intraclass correlation) was applied to the data obtained.
  • the intraclass correlation coefficient was determined as 0.7, using SPSS 19 (Statistical Package for the Social Sciences; IBM Corporation), which is considered strong agreement.
  • the epithelial/cancer and stromal compartments of cancer were separately analyzed using the Manual ROI Selection (Select Segments) segmentation tool, with a segmentation of 8.
  • the hematoxylin and immunohistological (IHC) threshold were set at 0.12 arbitrary units (a.u.) and 0.03 a.u., respectively.
  • the IHC threshold was determined by identifying the lightest positively-stained nucleus in the sample set and using this value as the cutoff for positivity.
  • a Nucleus Morphology and Filter step was used to exclude objects mistakenly identified as nuclei. From the exported results, positive indices were computed per cell type per patient.
  • Cilia Score (Cilia + Ki67): Scoring percent ciliated cancer cells may under-represent the cilia-positive fraction of cancer cells because cilia are only expressed on cells in GO or early-Gl of the cell cycle. To correct for this Such experiments will add the percent cilia- positive cancer cells to the percent Ki67-positive cancer cells for each imaged region to obtain a cilia + Ki67 score.
  • Example VIII Example VIII.
  • NSCLC cell lines that staining determined were cilia-negative (NCI-H1975 and NCI-H2030) both had a ciliogenesis gene signature score of ⁇ 11 ( Figure 10B and IOC respectively). Therefore, "12" was set as a minimum cilia prediction cutoff, and this predicted that 38% of NSCLC cell lines are cilia- positive ( Figure IOC, horizontal dashed line).

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Abstract

La présente invention concerne des biomarqueurs pour la détection et la caractérisation de cellules cancéreuses liées à l'activité de la voie hedgehog (par exemple cancer du poumon non à petites cellules), ainsi que des utilisations associées de ces biomarqueurs.
PCT/US2017/023839 2016-03-23 2017-03-23 Biomarqueurs pour un cancer lié à l'activité de la voie hedgehog et utilisations associées de ces biomarqueurs Ceased WO2017165663A1 (fr)

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CN110618271A (zh) * 2019-09-29 2019-12-27 中国医学科学院肿瘤医院 非小细胞肺癌的预后预测方法
CN110714076A (zh) * 2018-07-13 2020-01-21 复旦大学 Arl14作为肿瘤标志物在制备预测肺腺癌预后及靶点药物中的用途
WO2020165370A1 (fr) * 2019-02-13 2020-08-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés et compositions pour sélectionner un traitement du cancer chez un sujet souffrant d'un cancer
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ES2902730A1 (es) * 2020-09-29 2022-03-29 Servicio De Salud De Castilla La Mancha Sescam Metodo para el diagnostico y/o pronostico de esofagitis eosinofilica en saliva
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CN110714076A (zh) * 2018-07-13 2020-01-21 复旦大学 Arl14作为肿瘤标志物在制备预测肺腺癌预后及靶点药物中的用途
CN110714076B (zh) * 2018-07-13 2022-11-04 复旦大学 Arl14作为肿瘤标志物在制备预测肺腺癌预后及靶点药物中的用途
WO2020165370A1 (fr) * 2019-02-13 2020-08-20 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédés et compositions pour sélectionner un traitement du cancer chez un sujet souffrant d'un cancer
US20220098674A1 (en) * 2019-02-13 2022-03-31 Inserm (Institut National De La Santé Et Dr La Recherch Médicale) Methods and compositions for selecting a cancer treatment in a subject suffering from cancer
CN110618271A (zh) * 2019-09-29 2019-12-27 中国医学科学院肿瘤医院 非小细胞肺癌的预后预测方法
US20210259954A1 (en) * 2019-11-27 2021-08-26 Chapman University Cilia-targeting nanoparticles
JP2022012466A (ja) * 2020-07-01 2022-01-17 学校法人関西医科大学 尿路系上皮腫瘍関連病変を検出するためのバイオマーカー
JP7560850B2 (ja) 2020-07-01 2024-10-03 学校法人関西医科大学 尿路系上皮腫瘍関連病変を検出するためのバイオマーカー
ES2902730A1 (es) * 2020-09-29 2022-03-29 Servicio De Salud De Castilla La Mancha Sescam Metodo para el diagnostico y/o pronostico de esofagitis eosinofilica en saliva
WO2023101846A3 (fr) * 2021-11-30 2023-09-28 Gradalis, Inc. Procédés de traitement de la réponse à des cancers

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