WO2017165663A1

WO2017165663A1 - Biomarkers for cancer associated with hedgehog pathway activity and related uses thereof

Info

Publication number: WO2017165663A1
Application number: PCT/US2017/023839
Authority: WO
Inventors: Kimberly MCDERMOTT; Nadia Bassam HASSOUNAH
Original assignee: University of Arizona
Current assignee: University of Arizona
Priority date: 2016-03-23
Filing date: 2017-03-23
Publication date: 2017-09-28
Anticipated expiration: 2018-09-23

Abstract

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.

Description

BIOMARKERS FOR CANCER ASSOCIATED WITH HEDGEHOG PATHWAY ACTIVITY AND RELATED USES THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of U. S. Provisional Application No. 62/312,262, filed March 23, 2016, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

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.

There remains an urgent need for effective therapeutic strategies to treat lung cancer.

SUMMARY OF THE INVENTION

The hedgehog (Hh) pathway drives tumor cell survival in many cancers, including

NSCLC. 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. However, 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.

In normal cells primary cilia are required for activation of the Hh pathway.

However, in cancer cells new data suggests that the Hh pathway can be activated via cilia- dependent or cilia- independent mechanisms (see, e.g., Hassounah, Ν.Β., 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

differentially depending on the absence or presence of Hh ligand (Fig. 1). In the absence of Hh, the Hh pathway stays in an Off state via processing of Gli transcription factors to the repressor (Gli^) form which blocks transcriptional activation of Hh genes (Fig. 1A). This is cilia-dependent through localization of Patched (Ptchl or Ptcl), a negative regulator of the pathway, to the ciliary membrane. In the presence of Hh, its receptor, Ptch, moves out of the cilium and Smo is phosphorylated and translocated into the cilium where it functions to promote Gli activation (Gli^A) (Fig. 1C) (see, e.g., Corbit, K.C., et al, 2005 Nature 437, 1018-1021 ; Milenkovic, L., et al, 2009 J Cell Biol 187, 365-374; Chen, Y., et al., 2011 PLoS Biol 9, el001083). In contrast, 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).

Experiments conducted during the course of developing embodiments for the present invention demonstrated that the primary cilia status of tumors correlates with Hh- targeted drug efficacy. Such experiments demonstrated that cancer cells activate the Hh- pathway through two distinct pathways, one that is cilia-dependent and one that is cilia- independent. Further experiments demonstrate that cilia are differentially expressed in Hh- activated NSCLC patient samples. Importantly, such experiments indicate that the relationship between the Hh- signaling pathway and cilia mechanistically influences the effectiveness of Hh-pathway drug inhibitors. Such experiments indicate that NSCLC cells that are positive for primary cilia will respond to the Hh-pathway inhibitors (e.g., Taladegib (LY2940680), Vismodegib).

Further experiments determined that individuals having cancer cells associated with 1) Hh pathway activity and 2) primary cilia expression have a unique biomarker gene expression profile or signature that can be used for predicting the presence or absence of primary cilia expression in cancer cells associated with Hh pathway activity, and as such, can be used for predicting responsiveness (e.g., favorable or unfavorable) to a Hh inhibitor treatment regimen for treating the cancer (e.g., NSCLC).

Accordingly, in certain embodiments, the present invention provides methods for selecting an individual having cancer cells associated with Hh pathway activity for treatment with a Hh inhibitor, comprising

a) obtaining a biological sample from the individual, wherein the biological sample comprises cancer cells,

b) determining a gene expression profile for one or more biomarker genes within the obtained cancer cells; and

c) administering to the individual a therapeutically effective amount of a Hh inhibitor if the determined biomarker gene expression profile is consistent with a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression, or not administering to the individual a therapeutically effective amount of a Hh inhibitor if the determined biomarker gene expression profile is inconsistent with a reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression.

Similarly, in certain embodiments, 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.

In certain embodiments, 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.

Such methods are not limited to specific biomarker genes for determining the gene expression profile. In some embodiments, 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.

In some embodiments, 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, CENPM, DDN, DLGAP5, DTL, E2F2, EXOl, FAM64A, GINS2, HELLS, HJURP, KIAA0101, KIF15, KIF20A, KIFC1, LRRc45, LRRC26, MCM10, MCM2, MCM4, MCM5, MCM8, MY019, NDC80, NMU, NRGN, NUDT1, PKMYT1, POLE, POLE2, PSMC3IP, RECQL4,

SLC39A10, SLC43A3, SPC24, SPC25, TK1, TOP2A, TYMS, UBE2C, XRCC3, GLI1, PTCH1, LRRC45, IFT43, WDR35, IFT122, TTC21B, IFT140, WDR19, TULP3, IFT20, RABL5, HSPBl l, IFT27, IFT46, IFT52, TRAF3IP1, IFT57, TTC30B, IFT74, IFT80, IFT81, IFT88, IFT172, KIF3A, KIF3B, KIFAP3, KIF17, DYNC2H1, DYNC2LI1, WDR34, DYNLT1, BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS12, RAB3IP, RAB8A, PCM1, PKD1, PKD2, PKHD1, AHI1, ARL13B, INPP5E, TMEM216, MKS1, TMEM67, CC2D2A, SDCCAG8, SEPT7, NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, EVC, EVC2, VHL, OFD1, STIL, and RPGR). In some embodiments, the one or more biomarker genes are selected from

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. In some embodiments, 1 biomarker gene is utilized. In some embodiments, 5 biomarker genes are utilized in determining the gene expression profile. In some embodiments, more than 10 (e.g., 10, 11, 12, 15, 20, 25, 50, 100, 1000, etc.) are utilized in determining the gene expression profile.

In some embodiments, 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 .

In such methods, 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.

In some embodiments, the Hh inhibitor is a cilia dependent Hh inhibitor.

In some embodiments, 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.

In some embodiments, 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). In some embodiments, the 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.

Additional examples of suitable 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

2007/054623, WO 2007/059157, WO 2007/120827, WO 2007/131201, WO 2008/070357, WO 2008/110611, WO 2008/112913, and WO 2008/131354.

Additional examples of suitable Hh inhibitors include, but are not limited to, BMS-

833923 (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.

2010/0093625); SANT-1 and SANT-2 (see, e.g., Rominger CM. et al., J. Pharmacol. Exp. Ther. 2009; 329(3):995-1005; l-piperazinyl-4-arylphthalazines or analogues thereof (see, e.g., Lucas B.S. et al., Bioorg. Med. Chem. Lett. 2010; 20(12):3618-22).

In some embodiments, 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. In some embodiments, the reference gene expression profile for cancer cells associated with Hh pathway activity and primary cilia expression is generated as described in Example VI.

In certain embodiments, 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.

In certain embodiments, 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.

In certain embodiments, such methods can be used for identifying new Hh inhibiting drugs in the treatment of a cancer associated with Hh pathway activity.

In certain embodiments, 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 cilia associated centrosome, and the antibodies against markers for activated Hh signaling indicates the presence of primary cilia expression in the cancer cells.

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. In some embodiments, the antibody against a ciliary axoneme i s anti-acetylated- tubulin. In some embodiments, the antibodies against a cilia associated centrosome is anti- gamma (Y)-tubulin. In some embodiments, the antibodies against markers for activated Hh signalling is anti-Ptchl antibody and/or anti-Glil antibody and/or anti-SMO antibody.

In certain embodiments, 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.

Similarly, in certain embodiments, 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. In such methods, 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.

In such methods, 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.

Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein. DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that primary cilia are both positive and negative regulators of the Hh pathway. A. Immunofluorescent staining of primary cilia (grey-scaled white arrows) with antibodies recognizing Acetylated-Tubulin (grey-scaled red, ciliary axoneme or cilium) and gamma-Tubulin (grey-scaled green, basal body). B. Cilia-dependent inhibition of the Hh pathway via processing of Gli^R (in the absence of Hh ligand). Gli^R inhibits non-canonical activation of Hh target genes. C. Cilia-dependent activation of the Hh pathway via Smo localization to the cilium and processing of Gli^A (in the presence of Hh ligand).

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. B. Human BCC tissues that went on to be treated with GDC-0449 were stained for markers of primary cilian and assigned a cilia score based on percentage of ciliated cells normalized to cell cycle (Ki67 index) (n=20). Best response was evaluated on tumor size evaluated following treatment and included: Progressive Disease (PD, >20% increase in tumor size), Stable Disease (SD, 0-30% decrease in tumor size), Partial Response (PR, 30-99% decrease) and Complete Response (CR, 100% decrease). *p<0.05

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

NSCLC patients.

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.

DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below:

The term "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. The term "treat," "treating," or "treatment" 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.

The term "therapeutically 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. Furthermore, 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.

The term "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. The term "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.

The term "responsiveness" or "responsive" when used in reference to a treatment refer to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, e.g., NSCLC, being treated. For example, the term "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. In certain embodiments, 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%.

The term "effective patient response" refers to an increase in the therapeutic benefit to a patient in treating a disease, e.g., NSCLC. In certain embodiments, 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.

The term "likelihood" refers to an increase in the probability of an event. The term "likelihood" 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.

The term "predict" generally means to determine or tell in advance. When used to "predict" the effectiveness of the treatment of a disease (e.g., NSCLC), for example, 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.

The terms "determining", "measuring", "evaluating", "assessing" and "assaying" are used interchangeably herein to refer to a form of measurement, including 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.

The term "biological sample" as used herein 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. In certain embodiments, biological samples include, but are not limited to, whole blood, partially purified blood, PBMCs, tissue biopsies, and the like.

DETAILED DESCRIPTION OF THE INVENTION

Non-small cell lung cancers (NSCLC) remain the leading cause of cancer deaths in men and women worldwide (s ee, Ferlay, J., et al, 2008 Int J Cancer 127, 2893-2917).

While treatment advances over the past few decades are making small gains in progression free survival, NSCLC remains a largely terminal disease. As such, there is a critical need to identify game changing drug targets. Abnormal activation of the oncogenic Hedgehog (Hh) signaling pathway is common in solid tumors, including NSCLC. Activation of the Hh pathway increases proliferation and activates pro-survival signals. In preclinical studies, blockade of the Hh pathway has been shown to decrease tumor growth and therefore, is emerging as a promising drug target for cancer treatment (see, e.g., Chang, A.L., et al., (2014) J Am Acad Dermatol 70, 60-69). The effectiveness of these compounds in preclinical models was highly promising. However, when tested in clinical trials across many cancer types, the results were highly inconsistent and ranged from complete response in some patients and progression on agent in others; results that support an underlying mechanism of individual patient response heterogeneity. Classic measures of Hh-pathway activation did not, however, prove predictive in correlative biomarker studies. Thus, a clear need exists to establish the mechanism(s) that underlie Hh-targeted drug response/resistance for the purpose of developing tumor biomarkers to optimize patient selection for use of Hh- inhibitors as well as for the long-term goal of developing next generation Hh pathway inhibitors.

Experiments conducted during the course of developing embodiments for the present invention demonstrated that primary cilia expression levels on an aggressive form of basal cell carcinoma (BCC) predicts patient response to Hh inhibitors. The primary cilium is a cellular organelle shown to regulate the Hh pathway in response to extracellular cues. While cilia are required for activation of the Hh pathway in normal cells, cancer cells activate the Hh pathway via cilia-dependent or -independent mechanisms suggesting a deregulation of the Hh/cilia interaction in some but not all cancers (see, e.g., Hassounah, N.B., et al., 2012 Clin Cancer Res 18, 2429-2435). For aggressive BCC, it was shown that cilia (present or absent) profoundly influences tumor response to Hh pathway inhibitors. Specifically, the presence of cilia (normalized to cell cycle) was highly correlated with tumor regression and clinical remission in a subset of BCC patient receiving Vismodegib (GDC-0449). This contrasted with the failure of tumor Hh activity status to predict patient response to Hh inhibitors. Importantly, such experiments conducted to understand the mechanism of Hh-inhibitor responsiveness suggests a dependence of Vismodegib anti-tumor activity on the presence of cilia. Furthermore, this dependence on cilia is mediated through a post-translational processing of Gli into a transcriptional repressor of tumor growth.

Accordingly, methods, systems, compositions, arrays, kits, reagents, computers software, and reports are provided herein for use in analyzing one or more of the biomarkers (e.g., presence of primary cilia expression in cancer cells) or biomarker genes (e.g., gene expression profiles) disclosed herein.

In one aspect, disclosed herein are methods for stratifying an individual having a cancer associated with Hh pathway activity (e.g., NSCLC) for treatment, based on the presence or absence of a specific gene expression profile for one or more biomarker genes selected from Figs. 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, GINS2, HELLS, HJURP, KIAA0101, KIF15, KIF20A, KIFC1, LRRc45, LRRC26, MCM10, MCM2, MCM4, MCM5, MCM8, MY019, NDC80, NMU, NRGN, NUDT1, PKMYT1, POLE, POLE2, PSMC3IP, RECQL4, SLC39A10, SLC43A3, SPC24, SPC25, TK1, TOP2A, TYMS, UBE2C, XRCC3, GLI1, PTCH1, LRRC45, IFT43, WDR35, IFT122, TTC21B, IFT140, WDR19, TULP3, IFT20, RABL5, HSPBl l, IFT27, IFT46, IFT52, TRAF3IP1, IFT57, TTC30B, IFT74, IFT80, IFT81, IFT88, IFT172, KIF3A, KIF3B, KIFAP3, KIF17, DYNC2H1, DYNC2LI1, WDR34, DYNLT1, BBSl, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS12, RAB3IP, RAB8A, PCM1, PKDl, PKD2, PKHDl, AHI1, ARL13B, INPP5E, TMEM216, MKS1, TMEM67, CC2D2A, SDCCAG8, SEPT7, NPHP1, INVS, NPHP3, NPHP4, IQCBl, CEP290, GLIS2, RPGRIP1L, NEK8, EVC, EVC2, VHL, OFD1, STIL, and RPGR).

In one aspect, disclosed herein are methods for stratifying an individual having a cancer associated with Hh pathway activity (e.g., NSCLC) for treatment, based on the presence or absence of a specific gene expression profile for one or more biomarker genes selected from TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBSl, PHKHD1, RABL5, HSPBl l, PKDl, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCBl (see, e.g., Example VI). In some embodiments, 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.

In some cases, 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. In some cases, 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). In some cases, 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). In other cases, 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. In other cases, 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).

In one aspect, disclosed herein are methods for stratifying an individual having a cancer associated with Hh pathway activity (e.g., NSCLC) for treatment, based on the presence or absence of primary cilia expression detected in obtained cancer cells from the individual. In some embodiments, 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). In some cases, 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). In some cases, the absence of primary cilia expression in such obtained cancer cells indicate 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). In other cases, 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.

Also disclosed herein are systems for using biomarkers (e.g., primary cilia expression in cancer cells) or 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),

Vismodegib)).

In some cases, 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. In some embodiments, such 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. In some cases, the results are compiled into a report format for sending to a user.

Further disclosed herein are kits and arrays for using biomarkers (e.g., primary cilia expression in cancer cells) or biomarker gene expression profiles disclosed herein for use with the methods and systems disclosed above.

In some embodiments, 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).

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

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, and pancreatic cancer.

Also disclosed herein are methods of using 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.

In some embodiments, the treatment comprises administration of a Hh inhibitor. Such treatment is not limited to a particular type or kind of a Hh inhibitor. In some embodiments, 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. In some embodiments, 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).

Methods for determining a gene expression profile are well known in the art.

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.

As disclosed herein, 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. By "determining the modification(s)" is intended to determine a mutation within the biomarker gene or a biomarker protein. As used herein,

"modification" and "mutation" are used interchangeably. The term "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. By "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.

In certain aspects, 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. In one

embodiments, 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.

In some embodiments, 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.

In other embodiments, the determining the modification, expression, or presence of one or more biomarkers carried out by a diagnostic imaging technique.

In still other embodiments, the determining the modification, expression, or presence of one or more biomarkers carried out by a detectable solid substrate. In one embodiment, the 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,

immunohistochemistry, nucleic acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In some embodiments, 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. In some embodiments, detection of biomarkers is accomplished by ELISA. In some embodiments, detection of biomarkers is accomplished by electrochemiluminescence (ECL).

In some embodiments, 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).

Additionally, large numbers of 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.

Thus, in some embodiments, the detection of a biomarker or other protein of interest is assayed at the nucleic acid level using nucleic acid probes. The term "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.

For example, 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.

In one embodiment, 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. In an alternative embodiment, 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. USA 86: 1 173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6: 1 197), rolling circle replication (U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, biomarker expression is assessed by quantitative fluorogenic RT- PCR (i.e., the TaqManO System). 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.

In some embodiments, 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.

6,040, 138, 5,800,992, 6,020, 135, 6,033,860, 6,344,316, and U. S. Pat. Application

20120208706. 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

DiscoveryMAP® 250+ v. 2.0 from Myraid RBM.

Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U. S. Pat. No. 5,384,261. In some embodiments, an array is fabricated on a surface of virtually any shape or even a multiplicity of surfaces. In some embodiments, an array is a planar array surface. In some embodiments, 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. In some embodiments, 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. Thus, in some embodiments, 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. In some embodiments, labeled antibodies, binding portions thereof, or other binding partners are used. The word "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. In some embodiments, 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. Further, 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. Examples of 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. For example, enzymes and chromophoric molecules are conjugated to the antibodies by means of coupling agents, such as dialdehydes, carbodiimides, dimaleimides, and the like. Alternatively, conjugation occurs through a ligand-receptor pair. Examples of suitable ligand-receptor pairs are biotin- avidin or biotin-streptavidin, and antibody-antigen. In certain embodiments, expression or presence of one or more biomarkers or other proteins of interest within a biological sample, for example, a sample of bodily fluid, 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. Thus, the detection assays involve steps such as, but not limited to, immunoblotting, immunodiffusion, Immunoelectrophoresis, or immunoprecipitation.

In some embodiments, 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). In some embodiments, the sample is obtained from cells of NSCLC, basal cell carcinoma, pancreatic cancer, medulloblastoma, colon cancer, breast cancer, and/or bladder cancer.

In some embodiments, 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. In particular embodiments, the sample is a blood serum sample. In particular embodiments, the sample is from a fluid or tissue that is part of, or associated with, the lymphatic system or circulatory system. In some embodiments, the sample is a blood sample that is a venous, arterial, peripheral, tissue, cord blood sample. In particular embodiments, the sample is a blood cell sample containing one or more peripheral blood mononuclear cells (PBMCs). In some embodiments, the sample contains one or more circulating tumor cells (CTCs). In some embodiments, the sample contains one or more disseminated tumor cells (DTC, e.g., in a bone marrow aspirate sample).

In some embodiments, 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. Typically, for collection of a blood sample, an anti-coagulation agent (e.g., EDTA, or citrate and heparin or CPD (citrate, phosphate, dextrose) or comparable substances) is added to the sample to prevent coagulation of the blood. In some examples, the blood sample is collected in a collection tube that contains an amount of EDTA to prevent coagulation of the blood sample.

In some embodiments, 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.

In some embodiments, 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). For example, 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. In particular examples, 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. In some embodiments, the patient is administered a Hh inhibitor and one or more additional therapeutic agents.

In some embodiments, a Hh inhibitor is administered in combination with an additional therapeutic agent for the treatment of a cancer associated with Hh pathway activity. In some embodiments, 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).

In certain embodiments, 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). In some embodiments, 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). In a particular embodiment, the additional therapeutic agent(s) is an anticancer agent.

A number of suitable 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; 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. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for coadministration with Hh inhibitors are known to those skilled in the art.

In certain embodiments, 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. , flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); 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.

In still other embodiments, the 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); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g. , carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)); and 5) triazenes (e.g., dacarbazine (DTIC;

dimethyltriazenoimid-azolecarboxamide).

In some embodiments, 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)).

In still further embodiments, 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

(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;

MIH)); 10) adrenocortical suppressants (e.g., mitotane (ο,ρ'-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g. , prednisone); 12) progestins (e.g. , hydroxy progesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g. , diethylstilbestrol and ethinyl estradiol); 14) 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. For example, 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

(5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine)

Carboplatin Paraplatin Bristol-Myers Squibb

(platinum, diammine [1 ,1-cyclobutanedicarboxylato(2-)-0,

0']-,(SP-4-2))

Carmustine BCNU, BiCNU Bristol-Myers Squibb

(1 ,3-bis(2-chloroethyl)-1 -nitrosourea)

Carmustine with Polifeprosan 20 Implant Gliadel Wafer Guilford

Pharmaceuticals, Inc., Baltimore, MD

Celecoxib Celebrex Searle

(as 4-[5-(4-methylphenyl)-3- (trifluoromethyh-1 H-pyrazol-1 - Pharmaceuticals, yi] England

benzenesulfonamide)

Chlorambucil Leukeran GlaxoSmithKline

(4-[bis(2chlorethyl)amino]benzenebutanoic acid)

Cisplatin Platinol Bristol-Myers Squibb (PtCI₂H_eN₂)

Cladribine Leustatin, 2-CdA R.W. Johnson

(2-chloro-2'-deoxy-b-D-adenosine) Pharmaceutical

Research Institute, Raritan, NJ

Cyclophosphamide Cytoxan, Neosar Bristol-Myers Squibb

(2-[bis(2-chloroethyl)amino] tetrahydro-2H-13,2- oxazaphosphorine 2-oxide monohydrate)

Cytarabine Cytosar-U Pharmacia & Upjohn

(1-b-D-Arabinofuranosylcytosine, C₉H₁₃N₃O₅) Company cytarabine liposomal DepoCyt Skye

Pharmaceuticals, Inc., San Diego, CA

Dacarbazine DTIC-Dome Bayer AG,

(5-(3,3-dimethyl-l-triazeno)-imidazole-4-carboxamide Leverkusen, Germany (DTIC))

Dactinomycin, actinomycin D Cosmegen Merck

(actinomycin produced by Streptomyces parvullus,

Darbepoetin alfa Aranesp Amgen, Inc., (recombinant peptide) Thousand Oaks, CA daunorubicin liposomal DanuoXome Nexstar ((8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Pharmaceuticals, Inc., hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,1 1 -trihydroxy- Boulder, CO

1-methoxy-5,12-naphthacenedione hydrochloride)

Daunorubicin HCI, daunomycin Cerubidine Wyeth Ayerst,

((1 S ,3 S )-3-Acetyl-1 , 2,3,4,6, 1 1-hexahydro-3,5,12- Madison, NJ trihydroxy-10-methoxy-6,11 -dioxo-1 -naphthacenyl 3-amino-

2,3,6-trideoxy-(alpha)-L- lyxo -hexopyranoside

hydrochloride)

Denileukin diftitox Ontak Seragen, Inc., (recombinant peptide) Hopkinton, MA

Dexrazoxane Zinecard Pharmacia & Upjohn

((S)-4,4'-(1 -methyl-1 ,2-ethanediyl)bis-2,6-piperazinedione) Company

Docetaxel Taxotere Aventis

((2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, Pharmaceuticals, Inc., 13-ester with 5b-20-epoxy-12a,4,7b,10b,13a- Bridgewater, NJ hexahydroxytax- 1 1 -en-9-one 4-acetate 2-benzoate,

trihydrate)

Doxorubicin HCI Adriamycin, Pharmacia & Upjohn

(8S,10S)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo- Rubex Company hexopyranosyl)oxy] -8-glycolyl-7,8,9,10-tetrahydro-6,8,1 1 - trihydroxy-1 -methoxy-5,12-naphthacenedione

hydrochloride)

doxorubicin Adriamycin PFS Pharmacia & Upjohn

Intravenous Company injection

doxorubicin liposomal Doxil Sequus

Pharmaceuticals, Inc., Menlo park, CA dromostanolone propionate Dromostanolone Eli Lilly & Company,

(17b-Hydroxy-2a-methyl-5a-androstan-3-one propionate) Indianapolis, IN dromostanolone propionate Masterone Syntex, Corp., Palo injection Alto, CA

Elliott's B Solution Elliott's B Solution Orphan Medical, Inc

Epirubicin Ellence Pharmacia & Upjohn

((8S-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-arabino- Company hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,1 1 -trihydroxy- 8- (hydroxyacetyl)-1 -methoxy-5,12-naphthacenedione

hydrochloride)

Epoetin alfa Epogen Amgen, Inc (recombinant peptide)

Estramustine Emcyt Pharmacia & Upjohn

(estra-1 ,3,5(10)-triene-3,17-diol(17(beta))-, 3-[bis(2- Company chloroethyl)carbamate] 17-(dihydrogen phosphate),

disodium salt, monohydrate, or estradiol 3-[bis(2- chloroethyhcarbamate] 17-(dihydrogen phosphate),

disodium salt, monohydrate)

Etoposide phosphate Etopophos Bristol-Myers Squibb

(4'-Demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene- (beta)-D-glucopyranoside], 4'-(dihydrogen phosphate))

etoposide, VP-16 Vepesid Bristol-Myers Squibb

(4'-demethylepipodophyllotoxin 9-[4,6-0-(R)-ethylidene- (beta)-D-glucopyranoside])

Exemestane Aromasin Pharmacia & Upjohn

(6-methylenandrosta-1 ,4-diene-3, 17-dione) Company

Filgrastim Neupogen Amgen, Inc

(r-metHuG-CSF)

floxuridine (intraarterial) FUDR Roche

(2'-deoxy-5-fluorouridine)

Fludarabine Fludara Berlex Laboratories,

(fluorinated nucleotide analog of the antiviral agent Inc., Cedar Knolls, NJ vidarabine, 9-b -D-arabinofuranosyladenine (ara-A))

Fluorouracil, 5-FU Adrucil ICN Pharmaceuticals,

(5-fluoro-2,4(1 H,3H)-pyrimidinedione) Inc., Humacao, Puerto

Rico

Fulvestrant Faslodex IPR Pharmaceuticals,

(7-alpha-[9-(4,4,5,5,5-penta fluoropentylsulphinyl) Guayama, Puerto nonyl]estra-1 ,3,5-(10)- triene-3,17-beta-diol) Rico

Gemcitabine Gemzar Eli Lilly

(2'-deoxy-2', 2'-difluorocytidine monohydrochloride (b- isomer))

Gemtuzumab Ozogamicin Mylotarg Wyeth Ayerst (anti-CD33 hP67.6)

Goserelin acetate Zoladex Implant AstraZeneca

Pharmaceuticals

Hydroxyurea Hydrea Bristol-Myers Squibb

Ibritumomab Tiuxetan Zevalin Biogen IDEC, Inc.,

(immunoconjugate resulting from a thiourea covalent bond Cambridge MA between the monoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3- (p-isothiocyanatophenyl)- propyl]-[N-[2- bis(carboxymethyl)amino]-2-(methyl) -ethyl]glycine)

Idarubicin Idamycin Pharmacia & Upjohn

(5, 12-Naphthacenedione, 9-acetyl-7-[(3-amino-2,3,6- Company trideoxy-(alpha)-L- lyxo -hexopyranosyl)oxy]-7,8,9,10- tetrahydro-6,9,11 -trihydroxyhydrochloride, (7S- cis ))

Ifosfamide IFEX Bristol-Myers Squibb

(3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H- 1 ,3,2-oxazaphosphorine 2-oxide)

Imatinib Mesilate Gleevec Novartis AG, Basel,

(4-[(4-Methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3- Switzerland pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide

methanesulfonate)

Interferon alfa-2a Roferon-A Hoffmann-La Roche, (recombinant peptide) Inc., Nutley, NJ

Interferon alfa-2b Intron A Schering AG, Berlin, (recombinant peptide) (Lyophilized Germany

Betaseron)

Irinotecan HCI Camptosar Pharmacia & Upjohn

((4S)-4,1 1 -diethyl-4-hydroxy-9-[(4- piperi- Company dinopiperidino)carbonyloxy]-1 H-pyrano[3', 4': 6,7]

indolizino[1 ,2-b] quinoline-3,14(4H, 12H) dione

hydrochloride trihydrate)

Letrozole Femara Novartis

(4,4'-(1 H-1 ,2,4 -Triazol-1-ylmethylene) dibenzonitrile)

Leucovorin Wellcovorin, Immunex, Corp.,

(L-Glutamic acid, N[4[[(2amino-5-formyl1 ,4,5,6,7,8 - Leucovorin Seattle, WA hexahydro4oxo6-pteridinyl)methyl]amino]benzoyl], calcium

salt (1 :1))

Levamisole HCI Ergamisol Janssen Research

((-)-( S)-2,3,5, 6-tetrahydro-6-phenylimidazo [2,1 -b] thiazole Foundation, Titusville, monohydrochloride Cii Hi₂N₂S-HCI) NJ

Lomustine CeeNU Bristol-Myers Squibb

(1-(2-chloro-ethyl)-3-cyclohexyl-1 -nitrosourea)

Meclorethamine, nitrogen mustard Mustargen Merck

(2-chloro-N-(2-chloroethyl)-N-methylethanamine

hydrochloride) Megestrol acetate Megace Bristol-Myers Squibb

17a( acetyloxy)- 6- methylpregna- 4,6- diene- 3,20- dione

Melphalan, L-PAM Alkeran GlaxoSmithKline

(4-[bis(2-chloroethyl) amino]-L-phenylalanine)

Mercaptopurine, 6-MP Purinethol GlaxoSmithKline

(1 ,7-dihydro-6 H -purine-6-thione monohydrate)

Mesna Mesnex Asta Medica

(sodium 2-mercaptoethane sulfonate)

Methotrexate Methotrexate Lederle Laboratories (N-[4-[[(2,4-diamino-6- pteridinyl)methyl]methylamino]benzoyl]-L-glutamic acid)

Methoxsalen Uvadex Therakos, Inc., Way

(9-methoxy-7H-furo[3,2-g][1 ]-benzopyran-7-one) Exton, Pa

Mitomycin C Mutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc.,

Dublin, CA

Mitotane Lysodren Bristol-Myers Squibb

(1 ,1 -dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane)

Mitoxantrone Novantrone Immunex Corporation

(1 ,4-dihydroxy-5,8-bis[[2- [(2- hydroxyethyl)amino]ethyl]amino]-9,10-anthracenedione

dihydrochloride)

Nandrolone phenpropionate Durabolin-50 Organon, Inc., West

Orange, NJ

Nofetumomab Verluma Boehringer Ingelheim

Pharma KG, Germany

Oprelvekin Neumega Genetics Institute, (IL-1 1) Inc., Alexandria, VA

Oxaliplatin Eloxatin Sanofi Synthelabo,

(cis-[(1 R,2R)-1 ,2-cyclohexanediamine-N,N'] [oxalato(2-)- Inc., NY, NY

Ο,Ο'] platinum)

Paclitaxel TAXOL Bristol-Myers Squibb

(5β, 20-Epoxy-1 ,2a, 4,7β, 10β, 13a-hexahydroxytax-1 1 -en- 9-one 4,10-diacetate 2- benzoate 13-ester with (2R, 3 S)- N-benzoyl-3-phenylisoserine)

Pamidronate Aredia Novartis

(phosphonic acid (3-amino-1 -hydroxypropylidene) bis-,

disodium salt, pentahydrate, (APD)) Pegademase Adagen Enzon

((monomethoxypolyethylene glycol succinimidyl) 1 1 - 17 - (Pegademase Pharmaceuticals, Inc., adenosine deaminase) Bovine) Bridgewater, NJ

Pegaspargase Oncaspar Enzon

(monomethoxypolyethylene glycol succinimidyl L- asparaginase)

Pegfilgrastim Neulasta Amgen, Inc

(covalent conjugate of recombinant methionyl human G- CSF (Filgrastim) and monomethoxypolyethylene glycol)

Pentostatin Nipent Parke-Davis

Pharmaceutical Co., Rockville, MD

Pipobroman Vercyte Abbott Laboratories,

Abbott Park, IL

Plicamycin, Mithramycin Mithracin Pfizer, Inc., NY, NY

(antibiotic produced by Streptomyces plicatus)

Porfimer sodium Photofrin QLT

Phototherapeutics, Inc., Vancouver, Canada

Procarbazine Matulane Sigma Tau

(N-isopropyl-p-(2-methylhydrazino)-p-toluamide Pharmaceuticals, Inc., monohydrochloride) Gaithersburg, MD

Quinacrine Atabrine Abbott Labs

(6-chloro-9-( 1 -methyl-4-diethyl-amine) butylamino-2- methoxyacridine)

Rasburicase Elitek Sanofi-Synthelabo, (recombinant peptide) Inc.,

Rituximab Rituxan Genentech, Inc.,

(recombinant anti-CD20 antibody) South San Francisco,

CA

Sargramostim Prokine Immunex Corp (recombinant peptide)

Streptozocin Zanosar Pharmacia & Upjohn

(streptozocin 2 -deoxy - 2 - Company

[[(methylnitrosoamino)carbonyl]amino] - a(and b ) - D - glucopyranose and 220 mg citric acid anhydrous)

Talc Sclerosol Bryan, Corp., Woburn,

(Mg₃Si₄O₁₀ (OH)₂) MA Tamoxifen Nolvadex AstraZeneca

((Z)2-[4-(1 ,2-diphenyl-1 -butenyl) phenoxy]-N, N- Pharmaceuticals dimethylethanamine 2-hydroxy-1 ,2,3- propanetricarboxylate (1 :1))

Temozolomide Temodar Schering

(3,4-dihydro-3-methyl-4-oxoimidazo[5,1 -d]-as-tetrazine-8- carboxamide)

teniposide, VM-26 Vumon Bristol-Myers Squibb

(4'-demethylepipodophyllotoxin 9-[4,6-0-(R)-2- thenylidene- (beta)-D-glucopyranoside])

Testolactone Teslac Bristol-Myers Squibb

(13-hydroxy-3-oxo-13,17-secoandrosta-1 ,4-dien-17-oic

acid [dgr ]-lactone)

Thioguanine, 6-TG Thioguanine GlaxoSmith Kline

(2-amino-1 ,7-dihydro-6 H - purine-6-thione)

Thiotepa Thioplex Immunex Corporation

(Aziridine, 1 ,1 ',1 "-phosphinothioylidynetris-, or Tris (1- aziridinyl) phosphine sulfide)

Topotecan HCI Hycamtin GlaxoSmithKline

((S)-10-[(dimethylamino) methyl]-4-ethyl-4,9-dihydroxy-1 H- pyrano[3', 4': 6,7] indolizino [1 ,2-b] quinoline-3,14- (41-1,12H)-dione monohydrochloride)

Toremifene Fareston Roberts

(2-(p-[(Z)-4-chloro-1 ,2-diphenyl-1-butenyl]-phenoxy)-N,N- Pharmaceutical Corp., dimethylethylamine citrate (1 :1)) Eatontown, NJ

Tositumomab, I 131 Tositumomab Bexxar Corixa Corp., Seattle, (recombinant murine immunotherapeutic monoclonal lgG_2a WA

lambda anti-CD20 antibody (I 131 is a

radioimmunotherapeutic antibody))

Trastuzumab Herceptin Genentech, Inc

(recombinant monoclonal IgGi kappa anti-HER2 antibody)

Tretinoin, ATRA Vesanoid Roche

(all-trans retinoic acid)

Uracil Mustard Uracil Mustard Roberts Labs

Capsules

Valrubicin, N-trifluoroacetyladriamycin-14-valerate Valstar Anthra --> Medeva ((2S-cis)-2- [1 ,2,3,4,6,11 -hexahydro-2, 5, 12-trihydroxy-7

methoxy-6,1 1 -dioxo-[[4 2,3,6-trideoxy-3- [(trifluoroacetyl)- amino-a-L-/yxo-hexopyranosyl]oxyl]-2-naphthacenyl]-2- oxoethyl pentanoate)

Vinblastine, Leurocristine Velban Eli Lilly

^eHseN^o-HzSO^

Vincristine Oncovin Eli Lilly

^eHseN^o-HzSO^

Vinorelbine Navelbine GlaxoSmithKline

(3' ,4'-didehydro-4'-deoxy-C'-norvincaleukoblastine [R- (R*,R*)-2,3-dihydroxybutanedioate (1 :2)(salt)])

Zoledronate, Zoledronic acid Zometa Novartis

((1-Hydroxy-2-imidazol-1 -yl-phosphonoethyl) phosphonic

acid monohydrate)

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, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, efl ornithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hul4.18-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-12, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafamib, luniliximab, mafosfamide, MB07133, MDX- 010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafin, MS-275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS- 9, 06-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774, panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS- 341, PSC 833, PXD101, pyrazoloacridine, Rl 15777, RADOOl, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-l, S-8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-1, valproic acid, vinflunine,

VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.

For a more detailed description of 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. For example, the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered to the animal using a linear accelerator. In still other embodiments, 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, mercaptopurine, methotrexate, fiuorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and the like), radioprotectors (e.g. , cysteamine, aminoalkyl dihydrogen phosphorothioates, amifostine (WR 2721), IL-1, IL-6, and the like). Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.

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. In one embodiment, the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.

In one embodiment, the total dose of radiation administered to an animal is about .01 Gray (Gy) to about 100 Gy. In another embodiment, 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) are administered over the course of treatment. While in some embodiments a complete dose of radiation can be administered over the course of one day, the total dose is ideally fractionated and

administered over several days. Desirably, 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). Accordingly, 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. If stretched over a period, in one embodiment, radiation is not administered every day, thereby allowing the animal to rest and the effects of the therapy to be realized. For example, 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. However, 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. For example, 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. Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks. These exemplary radiotherapy administration schedules are not intended, however, to limit the present invention.

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.

Disclosed herein, in certain embodiments, are systems of assessing an individual having a cancer associated with Hh pathway activity (e.g., NSCLC) for treatment 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.

In some embodiments, the systems and methods described herein include a digital processing device, or use of the same. In further embodiments, the digital processing device includes one or more hardware central processing units (CPU) that carry out the device's functions. In still further embodiments, the digital processing device further comprises an operating system configured to perform executable instructions. In some embodiments, the digital processing device is optionally connected to a computer network. In further embodiments, the digital processing device is optionally connected to the Intemet such that it accesses the World Wide Web. In still further embodiments, the digital processing device is optionally connected to a cloud computing infrastructure. In other embodiments, the digital processing device is optionally connected to an intranet. In other embodiments, the digital processing device is optionally connected to a data storage device.

In accordance with the description herein, 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. Those of skill in the art will recognize that many smartphones are suitable for use in the system described herein. Those of skill in the art will also recognize that select televisions, video players, and digital music players with optional computer network connectivity 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. EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. Example I.

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. All of the Smo inhibitors that are currently being evaluated in clinical trials (Vismodegib (Genentech), NVP-LDE225 (Novartis), IPI-926 (Infinity), and XL-139 (BMS/Exelixis)) are in this cilia-dependent class. Clinical trials utilizing Hh inhibitors are not considering cilia status of cancer cells as a criterion for determining the most effective Hh inhibitor.

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). Since being approved for treatment of BCC, Vismodegib has gone on to be investigated in 45 clinical trials involving over 18 different types of cancer. Note that NSCLC was not among the cancer types included in these clinical trials. However, several of these trials have been stopped due to poor clinical outcome. Long-term exposure to Vismodegib has been shown to result in resistant cancer cells that acquired novel mutations, which block the binding of Smo to Vismodegib (see, e.g., Rudin, CM., et al., 2009 N Engl J Med 361, 1173-1178). These results explain how some patients acquire resistance to Vismodegib over time; however, these types of Smo mutations have not been observed in primary cancers and hence do not explain why some Hh-active patients do not respond to initial treatment.

Experiments conducted during the course of developing embodiments for the present invention demonstrate that the reason Vismodegib is not effective in some cancers is because such cancers activate the Hh pathway in a cilia-independent manner and do not have a significant population of ciliated cancer cells.

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

(median: 58%) and progressive disease (median: 37%) (n=20; Fig. 3). This indicates that Smo inhibitors are cilia- dependent and suggests that BCC cancers with the highest percentage of ciliated cells are those that will benefit most from Vismodegib treatment. The significance of this observation is two-fold. First, this demonstrates that cilia status can serve as a predictive biomarker for Hh inhibitor efficacy. Second, these findings indicate that cilia status modulates tumor response to Hh inhibitors.

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. Such results directly address efficacy of Hh inhibitors in NSCLC; which, account for the greatest number of cancer-associated deaths worldwide each year. Based on this data, -36% of all NSCLC patients have primary cilia and would benefit from Hh-targeted therapy.

Example II.

It has recently been demonstrated that the 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). In this NSCLC cohort, Ptch (a Hh responsive gene indicating increased Hh signaling) correlated with decreased 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

(p=0.007) and Shh (p=0.001) expression (see, e.g., Raz G, et al, 76 Lung Cancer 2012, pages 191-196). This correlation suggests a dependence on Smo, and therefore cilia, for activation of the Hh pathway in NSCLC (Fig. 1C). Based on this data, it was hypothesized that a subset of NSCLC activate the Hh pathway via a cilia-dependent mechanism and that these patients are positive for primary cilia.

Glil, Ptchl, Smo and Shh markers alone are not sufficient to predict efficacy with the Hh-specific Smo inhibitor Vismodegib in clinical trials (Von Hoff, D.D., et al., 2009 N Engl J Med 361, 1164-1172). Demonstrating presence of primary cilia in these Hh-positive cases would provide further evidence of Smo activity in these patients and provide justification for clinical trials with the FDA approved Smo inhibitor Vismodegib. 25 NSCLC patient samples were stained for markers of primary cilia and it was found that 8 out of 25 NSCLC samples express primary cilia (Fig. 4A). 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.

Example III.

This example describes the experimental design for additional experiments related to Example II.

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. Tissue microarrays (TMA) with NSCLC samples used in a previously described Hh marker study (see, e.g., Raz G, et al, 76 Lung Cancer 2012, pages 191-196) have been obtained. The TMAs have duplicate 1mm cores representing a total of 245 NSCLC cases. The TMA includes 64.5% (n=158) adenocarcinoma, 26.9% (n=66) squamous cell carcinoma (SCC), and 21 of Other' types of NSCLC. The majority (77.6%) had stage I disease and the rest are 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.

R&D, Staining and Imaging: Serial sections of each TMA have been obtained. The first slide will be stained for H&E in order to annotate the cancer pathology. The H&E stained slide will be used to find the areas of interest to focus on in the next serial section, which will be stained for markers of primary cilia. Each tissue core of the digitally scanned TMA will be annotated and detailed histologic information provided. 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. 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).

Staining Analysis: 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 γ-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.

Based on preliminary data, it is expected that roughly 50% of the NSCLC samples will express markers of activated Hh signaling, resulting in 119 patient samples for assessment of percent cilia in Hh-active cases. Since the Spearman correlation coefficient is the Pearson correlation coefficient of the ranks, this sample size will allow us to detect a Spearman correlation of 0.25 or greater with 80% statistical power (assuming a two- sided alpha level of 0.05). Based on preliminary data demonstrating Smo and Shh correlates with downstream activity in the Hh pathway, such experiments are expected to find that patient samples that are positive for Smo, Shh, Glil and/or Ptchl activate the Hh pathway via primary cilia and therefore these patients will have a high cilia score. There is also preliminary data demonstrating that Hh-positive NSCLC has a high percentage of ciliated cancer cells further supporting the hypothesis. As described, such experiments will correlate cilia score with pathologic subtype (adenocarcinoma, squamous cell carcinoma and other), age at diagnosis, gender, smoking history, disease stage, relapse-free survival and 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.,

Chen, M., et al, 2010 Cancer Prev Res (Phila) 3, 1235-1245; Dai, J., et al., 2011 Pancreas 40, 233-236; Al-Halabi, H., et al., 2011 Acta Neuropathol 121, 229-239). In the NSCLC cohort, Ptchl correlated with decreased overall survival and decreased relapse-free survival (see, e.g., Yuan, Z., et al., 2007 Oncogene 26, 1046-1055). Therefore, if such experiments see that the Hh pathway is activated in these patients via a cilia-dependent mechanism it is also predict that the highest cilia scores will correlate with poor overall survival and decreased relapse-free survival. The opposite correlation is predicted if it is found that the Hh pathway is activated in patients via a cilia-independent mechanism. Results

demonstrating that 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.

Example IV.

In NSCLC, 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). In addition, downregulation of Hh signaling using small molecule inhibitors (Vismodegib, HhAntag, GANT61) or 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. These data are consistent with the hypothesis that some NSCLC require Hh signaling for tumorigenesis. Such results further suggests that patients with high Hh signaling would benefit from treatment with an Hh inhibitor. However, clinical trials using Vismodegib to treat BCC patients based on markers of high Hh pathway activity (Glil or Smo activating mutations) had mixed results indicating that these markers are not sufficient to predict which patients will respond to drug. Therefore, a better understanding of the mechanisms of Vismodegib response/resistance are urgently needed to better target this drug (and other Hh inhibitors) to the correct population of patients. Hh signaling can be activated in cancer cells in a cilia-dependent and cilia-independent manner. It was hypothesized that the Hh activating protein Smo is cilia-dependent and therefore Smo inhibitors such as Vismodegib will only be effective at treating cilia-positive cancer cells. Experiments conducted herein demonstrate that a subset of NSCLC are cilia-positive.

This experiment investigated if Hh-active NSCLC require cilia for targeted therapy with Vismodegib.

In vitro NSCLC cell lines. A NSCLC cell line (A549) that has high Hh signaling has been identified. Previously published studies demonstrate that A549 cells have reduced cell migration in vitro following treatment with the 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. Briefly, 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.

In vivo patient derived xenograft model. To evaluate the role of primary cilia and hedgehog inhibition in therapeutic treatment of NSCLC, it is proposed to perform a pre- clinical efficacy trial of the hedgehog inhibitor, Vismodegib alone or in combination with a standard of care treatment (cisplatin). For these studies, experiments will utilize two NSCLC patient derived tumor explants (PDX) derived from early stage lung

adenocarcinoma (LAC), chemo-radiation naive human patient LAC tumors that have been found to harbor either high or low scores for primary cilia expression (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. In addition, 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. Experiments have treated these LAC PDX models with cisplatin and it was found that A) the model is responsive to standard of care and B) this model is consistent across mice for tumor response and therefore useful for pre-clinical efficacy trials. While the LAC PDX model is responsive to standard of care, tumor growth is not completely inhibited. This response is consistent with what is observed in the clinic. For studies proposed in this application, LAC PDX tissue samples will be diced into slurry and mixed with growth factor depleted Matrigel. 6-8 week old SCID mice (male to female ratio = 1 : 1) will be injected in the flank with 200μ1 of the LAC PDX- Matrigel slurry. Upon development of palpable tumor, mice will undergo a baseline scan using the Pearl Impluse and then randomized into 4 therapy cohorts (n=10 mice each): a) untreated, b) Vismodegib (lOmg/kg) c) Vismodegib (25mg/kg) d)

Vismodegib (50mg/kg). Tumor volumes will be measured using calipers twice weekly. After four weeks of treatment, mice will undergo a final scan with the Pearl Impluse and sacrificed. Tumors will be collected for histological and biochemical analysis. Initial statistical analysis will focus upon evaluating the effects of Hh inhibition upon tumor growth via both comparison of the change in volume for individual tumors, as well as the change in tumor luminescence values (Relative light units-RLU) generated from the Pearl Impulse. Additional analysis of tumor tissue will focus upon comparisons between the ciliated and non-ciliated LAC PDXs, and will include analysis of pathology (H&E staining), Hh activity (GUI, Ptchl staining), proliferation (Ki-67 staining) and apoptosis (cleaved caspase 3 staining).

Cellular response in vitro: It is expected that Vismodegib will inhibit Hh signaling in cilia-positive cancers and not in cilia-negative cancers. Cilia-dependent inhibition of Hh signaling will result in reduced Glil, Gli2 and Ptch gene transcription following treatment with Vismodegib. It is also expect that treatment of the cilia-positive cells with Vismodegib will reduce cell viability. Results demonstrating that cilia-negative cancer cells are sensitive to a Smo inhibitor would suggest that Smo can activate the Hh pathway in a cilia-independent manner. This would be a novel finding and suggest that cancer cells can activate Smo without cilia. These findings would be unexpected but provide an exciting new avenue to study novel mechanisms of Hh activation in tumors.

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). In a colon tumor PDX model, response to Vismodegib was minimal at lOmg/Kg and response was saturated at 50mg/Kg (see, e.g., Wong, H., et al., 2011 Clin Cancer Res 77, 4682-4692). A similar dose response to Vismodegib in the LAC PDX model with a high cilia score is expected.

Example V.

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.

For the Gant61 Up and Gant61 Down list of genes used for the Hh Signature, the genes listed as provided in Figure 4 of cDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells. Shi, T. et. al. PLoS One, Oct 10, Vol 5, Issue 10, el3054. 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.

Example VI.

This example demonstrates gene expression signatures representing Hh pathway activity and ciliogenesis.

Evidence exists that primary cilia can be used as a companion diagnostic to predict

Hh-targeted drug efficacy in NSCLC. A bioinformatics approach was utilized to identify cancer patients that have expression of genes associated with the Hh pathway and with ciliogenesis.

First, patients with high levels of expression of Hh-target genes were identified. To accomplish this a 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

(Shi T, et al., PLoS ONE. 2010;5(10)). Note that 3 additional genes (GLI1, PTCH1,

LRRC45) that are known to go down when the Hh pathway is inhibited based on published literature were included. The level of expression of the GANT UP and GANT DOWN genes in a published NSCLC cancer microarray dataset (Hou J, Aerts J, et al., PLoS ONE.

2010;5(4):el0312) was analyzed. A Hh-target gene expression score was calculated for each patient using the following formula:

· 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).

Next, 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.

These 18 ciliogenesis genes were then utilized as the basis for the ciliogenesis gene expression signature. The level of expression of the 18 ciliogenesis genes in the same

NSCLC cancer microarray dataset described above was analyzed (Hou J, Aerts J, et al, PLoS

ONE. 2010;5(4):el0312). 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).

For healthy patients (normal) as well as patients with NSCLC (NSCLC types:

adenocarcinoma, squamous, and other) 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. As expected, this scatter-plot analysis demonstrated a positive correlation between ciliogenesis score and the Hh pathway activity score amongst patients with NSCLC (Pearson correlation coefficient = 0.80) (Fig. 6).

Furthermore, the scatter-plot analysis demonstrated that samples from healthy patients (normal) had lower ciliogenesis and Hh pathway activity scores than samples from patients with NSCLC.

This data strongly supports the use of the Hh pathway activity and ciliogenesis gene signatures to predict which NSCLC activate the Hh pathway via cilia-dependent mechanisms. It can further be utilized to identify patients that activate the Hh pathway via cilia- independent mechanisms.

Example VII.

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).

Human tissue specimens. All tissue sections used were formalin-fixed paraffin- embedded human tissue samples. Serial tissue sections were cut for each patient sample. The first serial section was stained for H&E and the entire tissue section was scanned with a 20x objective using the automated DMetrix slide scanner (DMetrix, Inc.). Digital images were annotated using the Eyepiece software (DMetrix, Inc.) by a certified pathologist for each of the areas of interest per tissue slide. Immunofluorescence. The tissue slide serial to the H&E-stained slide was used for co-staining of ciliary axoneme, and the centrosomes. 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

ChemMate Antibody Dilution Buffer at 1 : 1000. Primary antibodies were used against acetylated tubulin (mouse monocloncal IgG2B, Sigma, Cat# T7451, clone 6-11B-1), γ-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-IgG₂B (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). 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).

Confocal imaging. The tissue slide that was serially adjacent to the digitally scanned H&E slide was fluorescently stained for markers of ciliary axoneme, centrosomes, and Hoechst. The Leica TCS SP5 II laser scanning confocal microscope (Leica Microsystems) was used to image the fluorescently-stained slides. Areas of interest were found using a low- power magnification objective (lOx, 0.4 PI Apo) to visualize Hoechst counterstain on the fluorescently-stained slide and by referencing the annotated serially adjacent H&E that was digitally scanned. This allowed us to find the exact same area of interest that had been annotated by the pathologist on the H&E. Once the area of interest was found, 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 μηι, with a Z-step taken every 1 μηι. 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 μιτι, with a Z-step taken every 0.34 μηι (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.

Confocal 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.

Immunohistochemistry. Formalin-fixed paraffin-embedded tissue slides were stained with a similar protocol used for immunofiuorescently stained slides described above. Antigen retrieval was performed as described but with the Vector Antigen Unmasking Solution (1 : 106) (Vector Laboratories, Cat# H-3300), followed by quenching of endogenous peroxidase activity at room temperature for 20 minutes using hydrogen peroxide in methanol (0.3%). Slides were then washed with PBS (4 x 10 min), placed into Shandon Coverplates (Thermo Scientific, Cat# 72-110-017) and then into Sequenza Slide Racks (Thermo

Scientific, Cat# NC0263065). Slides were then blocked with 2.5% normal horse serum blocking buffer (Vector Laboratories, Cat# S-2012) for 20 minutes, and then with ChemMate Antibody Dilution Buffer (Ventana Medical Systems, Inc., Cat# ADB250) with goat serum (5%) (Invitrogen Corporation, Cat# 16210-064) for 45 minutes. All washes and blocking steps were at room temperature. Primary antibodies were diluted in the ChemMate Antibody Dilution Buffer. The following primary antibody was used: Ki67 (1 : 100, mouse monoclonal IgGi, Dako, Cat#M7240, clone MIB-1). Tissue slides with secondary antibody only were used as a negative control. Primary antibodies were incubated on tissues overnight at 4°C. Slides were then washed in PBS (3 x 10 min). A universal anti-rabbit, anti-mouse secondary antibody conjugated to peroxidase (ImmPress Universal Reagent, Vector Laboratories, Cat# MP-7500) was incubated on the tissue for 30 minutes, followed by a wash in PBS for 5 minutes. 3-amino-9-ethylcarbazole (AEC) with high sensitivity substrate chromagen (Dako, Cat# K3461) was use as the peroxidase substrate for Ki67 staining. AEC was incubated on slides for 7 minutes for Ki67 staining. Tissue slides were rinsed with distilled water for 5 minutes. 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).

Immunohistochemistry analysis. Whole slides stained for Ki67 were scanned using a 20x objective with the Biolmagene scanner (Ventana Medical Systems). The same locations used for primary cilia analysis were found by referencing the annotated H&E slide. These areas of interest were exported as TIFF files from the Dmetrix scan files, and as JPEG files from the Biolmagene scan files, and uploaded into Definiens Tissue Studio 3.0 Software (http://www.tissuestudio.com). Tissue Studio 3.0 Software was tested for absolute agreement with manual hand counts performed by two separate investigators. For each image, Tissue Studio 3.0 was used in conjunction to quantify the number of positive and negative cells/nuclei. 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.

For the Ki67 analysis with Definiens Tissue Studio, a modified Nuclei

(Positive/Negative) solution was used. 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. Interpretation of cilia + Ki67 score will follow these examples: 1.) cilia (-30%) + Ki67 (-70%) =100%, therefore all of the cancer cells have the ability to make cilia; 2.) cilia (-30%) + Ki67 (-30%) =60%, heterogeneity amongst the cancer cells that have the potential to make cilia; 3.) cilia (0%) + Ki67 (-50%) =50%, interpreted as a cilia-negative sample. Example VIII.

To determine if the ciliogenesis gene signature could be utilized to predict expression of primary cilia, experiments were conducted that utilized a publically available gene expression dataset of 47 NSCLC cell lines to analyze the ciliogenesis gene signature. This data set was provided by Genentech (Dataset: RNA-seq of 675 commonly used human cancer cell lines) is publicly available at the Expression Atlas.

Four of the NSCLC cell lines (NCI-H2073, NCI-H2228, NCI-H1975, and NIC- H2030) in this gene expression dataset were stained for markers of primary cilia. Based on immunocytochemistry for markers of primary cilia, two of the four NSCLC cell lines tested (NCI-H2228 and NCI-H2073) were positive for primary cilia expression (Figure 10A and 10B). Experiments analyzed the ciliogenesis gene signature in each of the 47 NSCLC cell lines and found that the two cilia-positive cell lines, NCI-H2228, and NCI-H2073, had a ciliogenesis gene signature >\2 (Figure IOC). The two 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).

Experiments also analyzed the Hh gene signature in each of the 47 NSCLC cell lines. Using >2 as a minimum cutoff for Hh-positive, the ciliogenesis and Hh signature scores suggest that 29% of NSCLC cell lines are both Hh-positive and Cilia-positive (Figure IOC, upper right quadrant). This is consistent with previous studies staining NSCLC patient tissue samples, which demonstrated that -30% are Hh-positive/Cilia-positive. These results strongly support the conclusion that the ciliogenesis gene signature is predictive of expression of primary cilia in NSCLC. All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention. INCORPORATION BY REFERENCE

The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A method for selecting an individual having cancer cells associated with Hh pathway activity for treatment with a Hh inhbitor, comprising:

2. The method of claim 1, wherein the one or more biomarker genes are selected from 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, CENPM, DDN, DLGAP5, DTL, E2F2, EXOl, FAM64A, GINS2, HELLS, HJURP, KIAAOlOl, KIF15, KIF20A, KIFCl, LRRc45, LRRC26, MCMIO, MCM2, MCM4, MCM5, MCM8, MY019, NDC80, NMU, NRGN, NUDT1, PKMYT1, POLE,

POLE2, PSMC3IP, RECQL4, SLC39A10, SLC43A3, SPC24, SPC25, TK1, TOP2A, TYMS, UBE2C, XRCC3, GLI1, PTCH1, LRRC45, IFT43, WDR35, IFT122, TTC21B, IFT140, WDR19, TULP3, IFT20, RABL5, HSPB11, IFT27, IFT46, IFT52, TRAF3IP1, IFT57, TTC30B, IFT74, IFT80, IFT81, IFT88, IFT172, KIF3A, KIF3B, KIFAP3, KIF17,

DYNC2H1, DYNC2LI1, WDR34, DYNLT1, BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS 12, RAB3IP, RAB8A, PCM1, PKD1, PKD2, PKHD1, AHI1, ARL13B, INPP5E, TMEM216, MKS1, TMEM67, CC2D2A, SDCCAG8, SEPT7, NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, EVC, EVC2, VHL, OFD1, STIL, and RPGR

3. The method of claim 1, wherein the one or more biomarker genes are selected from TMEM216, PCMl, IFT140, IFT122, TRIM32, MKKS, BBSl, PHKHDl, RABL5, HSPBl l, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1.

4. The method of claim 1, wherein the determining the gene expression profile involves quantifying mRNA expression alnd protein expression related to the specific biomarker genes within the obtained cancer cells.

5. The method of claim 1, wherein 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.

6. The method of claim 1, wherein the 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.

7. A method for treating a human subject having cancer cells 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.

8. The method of Claim 7, wherein the one or more biomarker genes are selected from 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, GINS2, HELLS, HJURP, KIAAOlOl, KIF15, KIF20A, KIFCl, LRRc45, LRRC26, MCMIO, MCM2, MCM4, MCM5, MCM8, MY019, NDC80, NMU, NRGN, NUDT1, PKMYT1, POLE, POLE2, PSMC3IP, RECQL4, SLC39A10, SLC43A3, SPC24, SPC25, TKl, TOP2A, TYMS, UBE2C, XRCC3, GLI1, PTCH1, LRRC45, IFT43, WDR35, IFT122, TTC21B, IFT140, WDR19, TULP3, IFT20, RABL5, HSPB11, IFT27, IFT46, IFT52, TRAF3IP1, IFT57, TTC30B, IFT74, IFT80, IFT81, IFT88, IFT172, KIF3A, KIF3B, KIFAP3, KIF17,

DYNC2H1, DYNC2LI1, WDR34, DYNLT1, BBSl, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS 12, RAB3IP, RAB8A, PCMl, PKD1, PKD2, PKHD1, AHI1, ARL13B, INPP5E, TMEM216, MKS1, TMEM67, CC2D2A, SDCCAG8, SEPT7, NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, EVC, EVC2, VHL, OFD1, STIL, and RPGR

9. The method of claim 7, wherein the one or more biomarker genes are selected from TMEM216, PCMl, IFT140, IFT122, TRIM32, MKKS, BBSl, PHKHDl, RABL5, HSPBl l, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1.

10. The method of claim 7, wherein the cancer cells associated with Hh pathway activity is selected from NSCLC cells, bladder cancer cells, basal cell carcinoma cells,

11. The method of claim 7, wherein the Hh inhibitor is selected from Taladegib

12. A method 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.

13. The method of claim 12, wherein the one or more biomarker genes are selected from 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, CENPM, DDN, DLGAP5, DTL, E2F2, EXOl, FAM64A, GINS2, HELLS, HJURP, KIAAOlOl, KIF15, KIF20A, KIFCl, LRRc45, LRRC26, MCMIO, MCM2, MCM4, MCM5, MCM8, MY019, NDC80, NMU, NRGN, NUDT1, PKMYT1, POLE, POLE2, PSMC3IP, RECQL4, SLC39A10, SLC43A3, SPC24, SPC25, TKl, TOP2A, TYMS, UBE2C, XRCC3, GLI1, PTCH1, LRRC45, IFT43, WDR35, IFT122, TTC21B, IFT140, WDR19, TULP3, IFT20, RABL5, HSPB11, IFT27, IFT46, IFT52, TRAF3IP1, IFT57, TTC30B, IFT74, IFT80, IFT81, IFT88, IFT172, KIF3A, KIF3B, KIFAP3, KIF17,

DYNC2H1, DYNC2LI1, WDR34, DYNLT1, BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS 12, RAB3IP, RAB8A, PCM1, PKD1, PKD2, PKHD1, AHI1, ARL13B, INPP5E, TMEM216, MKS1, TMEM67, CC2D2A, SDCCAG8, SEPT7, NPHP1, INVS, NPHP3, NPHP4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8, EVC, EVC2, VHL, OFD1, STIL, and RPGR.

14. The method of claim 12, wherein the one or more biomarker genes are selected from TMEM216, PCM1, IFT140, IFT122, TRIM32, MKKS, BBS1, PHKHD1, RABL5, HSPB11, PKD1, SDCCAG8, CDP290, STIL, IFT81, WDR34, TULP3, and IQCB1.

15. The method of claim 12, wherein the determining the gene expression profile involves quantifying mRNA expression alnd protein expression related to the specific biomarker genes within the obtained cancer cells.

16. A method for selecting an individual having cancer cells associated with Hh pathway activity for treatment with a Hh inhbitor, comprising:

b) detecting the presence or absence of primary cilia expression within the cancer cells, and

c) 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.

17. The method of Claim 16, wherein detecting the presence or absence of primary cilia expression within the cancer cells comprises:

1) exposing the cancer cells to

antibodies against a ciliary axoneme,

antibodies against a cilia associated centrosome,

antibodies against markers for activated Hh signaling, 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

2) 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 cilia associated centrosome, and the antibodies against markers for activated Hh signaling indicates the presence of primary cilia expression in the cancer cells.

18. The method of Claim 17, wherein the antibody against a ciliary axoneme i s anti- acetylated-tubulin.

19. The method of Claim 17, wherein the antibodies against a cilia associated centrosome is anti-gamma (y)-tubulin.

20. The method of Claim 17, wherein the antibodies against markers for activated Hh signalling is anti-Ptchl antibody and/or anti-Glil antibody and/or anti-SMO antibody.

21. The method of claim 16, wherein the cancer cells associated with Hh pathway activity are selected from NSCLC cells, bladder cancer cells, basal cell carcinoma cells,

22. The method of claim 16, wherein the 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.

23. A method for treating a human subject having cancer cells associated with Hh pathway activity, comprising the steps of:

a) obtaining a biological sample from the human subject, wherein the biological sample comprises cancer cells,

24. The method of Claim 23, wherein detecting the presence or absence of primary cilia expression within the cancer cells comprises:

1) exposing the cancer cells to

antibodies against a ciliary axoneme,

antibodies against a cilia associated centrosome,

25. The method of Claim 23, wherein the antibody against a ciliary axoneme i s anti- acetylated-tubulin.

26. The method of Claim 23, wherein the antibodies against a cilia associated centrosome is anti-gamma (y)-tubulin.

27. The method of claim 23, wherein the cancer cells associated with Hh pathway activity is selected from NSCLC cells, bladder cancer cells, basal cell carcinoma cells,

28. The method of claim 23, wherein the 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.

29. A method for determining the presence or absence of primary cilia expression in cancer cells associated with Hh pathway activity, comprising

a) obtaining a biological sample comprising cancer cells,

b) exposing the cancer cells to

antibodies against a ciliary axoneme,

antibodies against a cilia associated centrosome,

antibodies against markers for activated Hh signaling, 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 c) 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,

30. The method of Claim 29, wherein the antibody against a ciliary axoneme i s anti- acetylated-tubulin.

31. The method of Claim 29, wherein the antibodies against a cilia associated centrosome is anti-gamma (y)-tubulin.

32. The method of Claim 29, wherein the antibodies against markers for activated Hh signalling is anti-Ptchl antibody and/or anti-Glil antibody and/or anti-SMO antibody.