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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/170640 Al 13 December 2012 (13.12.2012) P O P C T

(51) International Patent Classification: (74) Agent: BOCK, Joel N. /Joel N. Bock, Reg. No. 36456/; C12Q 1/68 (2006.01) SNR DENTON US LLP, P.O. Box 061080, Wacker Drive Station, Willis Tower, Chicago, Illinois 60606 (US). (21) International Application Number: PCT/US20 12/04 1267 (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (22) Date: International Filing AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, 7 June 2012 (07.06.2012) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, (25) Filing Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, (26) Publication Language: English KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, (30) Priority Data: MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, 61/494,183 7 June 201 1 (07.06.201 1) US OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, (71) Applicant (for all designated States except US): THE TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK [US/US]; 412 Low Library, Mail (84) Designated States (unless otherwise indicated, for every Code 4308, 535 West 116th Street, New York, New York kind of regional protection available): ARIPO (BW, GH, 10027 (US). GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors; and TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (75) Inventors/Applicants (for US only): VUNDAVALLI, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Murty [US/US]; 560 Riverside Drive, Apartment 10H, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, New York, New York 10027 (US). XIE, Dongxu TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, [US/US]; 3475 Greystone Avenue, Apartment 2B, Bronx, ML, MR, NE, SN, TD, TG). New York 10463 (US). BHAGAT, Govind [US/US]; 250 Cabrini Boulevard, Apartment 7A, New York, New York Published: 10033 (US). — with international search report (Art. 21(3))

(54) Title: METHODS AND COMPOSITIONS FOR TRAIL-DRUG COMBINATION THERAPY (57) Abstract: Provided are methods for diagnosing conditions, such as cervical , T- hematologic malignancies, B-cell NHL or , susceptible to increased TRAIL-mediated apoptosis. Diagnosis can include the presence of one or more of an 8p chromosomal deletion, methylation-associated decoy receptor inactivation, or decreased decoy receptor expression. Also provided are methods of treatment and compound screening related to TRAIL-mediated apoptosis. Also provided are methods of treatment and compound screening related to cervical cancer, T-cell hematologic malignancies, B-cell NHL or breast cancer. METHODS AND COMPOSITIONS FOR TRAIL-DRUG COMBINATION THERAPY

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. Provisional Application Serial

No. 61/494,183, filed on June 7, 201 1, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number CA095647 awarded by National Institutes of Health. The government has certain rights in the invention.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Cervical Cancer (CC) affects over 0.5 million women worldwide. When invasive cancer is diagnosed the cure rate is low resulting in high mortality and the treatment response remains unpredictable.

Over 90% of invasive cervical cancer and Cervical Intraepithelial Neoplasia (CIN) contain human papilloma virus (HPV) DNA sequences. The high-risk HPV (hrHPV) type E6 and E7 interact with critical checkpoint genes p53 and pRb, respectively, interfere with the DNA repair mechanisms, as a consequence accelerate the accumulation of genetic alterations and immortalization (1). Conventional treatment of cervical cancer often employs using platinum based derivatives followed by radiation. But cervical cancer as a single diagnostic entity exhibits differences in clinical behavior and response to therapy, where advanced tumors remain unresponsive to chemo-radiotherapy. is used as the most effective agent in advanced stages of cervical cancer where most patients exhibit acquired resistance or progressive disease after initial response (2).

B-cell Non-Hodgkin (B-NHL) subtypes exhibit complex genetic changes that include characteristic chromosome translocations. Although recent molecular studies have identified certain prognostic markers, the low (<50%) progression- free survival achieved currently in high grade such as diffuse large-B-cell lymphoma (DLBCL) is primarily due to lack of understanding of the complex genetic and epigenetic lesions in these subtypes of B-NHL. Moreover, the yield of potential therapeutic targets discovered to date has been limited. Although overall survival has been significantly improved for B-cell lymphomas by the addition of rituximab, a large proportion of B-NHL patients still exhibit resistance to the existing treatments [34]. Therefore, there is a need for developing new therapies and biomarker of response to stratify patients that benefit specific treatments such as TRAIL combination drugs.

A majority of breast cancer patients exhibit resistance to either recombinant TRAIL or antibodies targeting TRAIL-R1/TRAIL-R2 despite the expression of death receptors on the cell surface [35]. The exact mechanisms of resistance in breast cancer patients are not well understood. Breast cancer is one of the top 3 that exhibit 8p21 .3 region deletions (at a proportion of 0.59) and exhibit frequent promoter methylation of either DcR1 or DcR2 genes [16, 36]. Although breast cancer as a group exhibits resistance to commonly used chemotherapy drugs such as and tomoxifen, patients with ER+ phenotype are the most resistant [37]. The underlying genetic determinants of TRAIL resistance/sensitivity in breast cancer cells are not known. We hypothesize that 8p deletion and decoy receptor inactivation may play a role in sensitizing breast cancer cells to TRAIL combination drug therapy. Therefore, these suboptimal outcomes of conventional treatments in breast cancer patients warrants an urgent need for the development of new therapies and identify biomarker to stratify patients that predict response to specific drug therapy.

T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL) and Peripheral T-cell lymphoma (PTCL) are a heterogeneous group of disorders committed to the T-cell lineage and represent distinct clinico-pathologic entities. Although chromosome abnormalities that generate fusion genes and alter genetic pathways e.g. INK4, NOTCH1, WT1, PTEN, PHF6 have been identified in a proportion of cases, their relationship with outcome is unclear [3-7]. Despite intensive risk-adopted chemotherapy protocols, the majority of T- cell leukemia/lymphoma (TCL) as a group exhibit resistance to therapy and shorter long- term disease-free survival with the exception of ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) that exhibit high cure rates with CHOP-like therapy. For example, studies using -containing regimens PTCL show only 32% cases with 5-year overall survival [5, 8]. The suboptimal outcome using intensive therapies for newly diagnosed TCL patients suggest that the current standard therapies are not effective and, therefore there is an urgent need for the development of new therapies. It has been recited in the art that "[m]uch effort has been devoted but failed to identify the biomarker(s) that can predict the sensitivity of human cancers to TRAIL-based therapies" and "it may be difficult if not impossible to identify the biomarkers that could predict the drug responsiveness in such genetically diversified [cancer]" (Bellail et al. 2009 4, 34-41, at 38).

Death receptor ligands, Fas and tumor necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL), play a role in cytotoxic T cell (CTC) and natural killer (NK) cell- mediated anti-tumor immunity. TCL cells acquire mechanisms to escape the host immune surveillance [9]. Despite the availability of novel genomic data and alterations of the associated genetic pathways, stratification of T-cell malignancies into subclasses responsive to specific treatment regimens is not feasible with agents currently being used [2, 10].

Tumor necrosis factor related super family (TNFRSF) of death receptors DR4 and DR5 express on the cell surface and are critical regulators of the extrinsic apoptotic pathway in TRAIL/AP02L-mediated cell death. Binding of TRAIL to their cognate DRs results in death inducing signaling complex (DISC) formation and DISC initiates activation of proteases caspase 8 and 10, thereby driving downstream effector caspase activation and apoptosis. TRAIL as a soluble zinc -coordinated homotrimeric triggers apoptosis only in cancer cells without affecting normal cells. Despite this specificity, many human cancers exhibit resistance to TRAIL [ 1 1, 12]. Since decoy receptors DcR1 and DcR2 compete with death receptors DR4 and DR5 in binding with TRAIL, over expression of DcR1 and DcR2 protects tumor cells from TRAIL-induced apoptosis. Efforts to identify differences in TRAIL responsiveness in tumor cells has identified decreased stimulation of TRAILR1 or R2 by DcRs, suppression of the intracellular signaling cascade genes, and post-translational modifications as important predictors [12, 22]. But it has been reported that TRAIL -resistance could not be predicted based on over expression of decoy receptors alone and the molecular mechanisms of resistance remain elusive [ 1 1-14].

Use of AP02L/TRAIL and TRAIL receptors as a single agent or in combination with other drugs is currently emerging as a promising cancer apoptosis-targeted therapy because of its unique tumor specificity [15]. It has been reported that diverse chemotherapeutic drugs or radiotherapy can exhibit synergistic tumor cell death when combined with recombinant TRAIL (rTRAIL) or agonistic anti-TRAILR mAbs [12]. Mechanisms that underlie this response are unknown. Previous studies on molecular events in TRAIL-mediated apoptosis focused on death receptors and other down-stream genes; but the role of genetic/epigenetic alterations in decoy receptors to TRAIL response has not been tested.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision of an effective TRAIL-drug combination therapy in patients carrying 8p deletion/decoy receptor inactivation in cervical cancer and lymphoid malignancies.

One aspect provides a method for determining responsiveness to TRAIL-induced apoptosis. In some embodiments, the method includes detecting an 8p chromosomal deletion in a sample. In some embodiments, the method includes detecting methylation of a promoter of at least one tumor necrosis factor related super family (TNFRSF) decoy receptor. In some embodiments, the method includes detecting expression level of at least one TNFRSF decoy receptor. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to presence of the 8p deletion. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to presence of methylation of the at least one decoy receptor. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to reduced expression levels of the at least one decoy receptor as compared to a control. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to two of (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control. In some embodiments, the method includes correlating increased TRAIL- induced apoptosis to (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, and (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

In some embodiments, the method includes detecting an 8p chromosomal deletion in a sample; and either or both of detecting methylation of a promoter of at least one tumor necrosis factor related super family (TNFRSF) decoy receptor or detecting expression level (e.g., decreased expression level) of at least one TNFRSF decoy receptor.

In some embodiments, the at least one decoy receptor comprises DcR1/TNFRSF10C. In some embodiments, the at least one decoy receptor comprises DcR2/TNFRSF10D. In some embodiments, the at least one decoy receptor comprises DcR1/TNFRSF10C and DcR2/TNFRSF10D.

In some embodiments, the method includes detecting methylation of a promoter of at least two decoy receptors. In some embodiments, the method includes detecting expression level of at least two decoy receptors. In some embodiments, the method includes detecting methylation of a promoter of at least two decoy receptors and detecting expression level of at least two decoy receptors. In some embodiments, the first decoy receptor comprises DcR1/TNFRSF10C and the second decoy receptor comprises DcR2/TNFRSF10D.

In some embodiments, the 8p deletion comprises a deletion at 8p12-p21 .3. In some embodiments, the 8p deletion comprises an 8.4 Mb minimal region of deletion (MRD) between 22,941-31,338 kb physical interval at 8p12-p21 .3.

In some embodiments, the sample comprises a tumor sample. In some embodiments, the sample comprises cervical cancer tumor sample, a T-cell leukemia/lymphoma tumor sample, a B-cell NHL tumor sample, or a breast cancer tumor sample. In some embodiments, the sample comprises a TRAIL-resistant tumor sample. In some embodiments, the sample is of a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is diagnosed with, or suspected of having, cervical cancer, T-cell leukemia/lymphoma, B-cell NHL, or breast cancer.

In some embodiments, the method includes selecting or modifying a treatment on the basis of detecting one, two, or three of: (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

In some embodiments, the method includes administering to the subject a therapeutically effective amount of a TRAIL agonist and an antineoplastic agent upon detecting one, two, or three of: (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

In some embodiments, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a TRAIL agonist, an antineoplastic agent, and a pharmaceutically acceptable carrier or excipient upon detecting one, two, or three of (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

In some embodiments, the TRAIL agonist is selected from the group consisting of Apo2L/TRAIL, HGS-ETR1 MAb, HGS-ETR2 MAb, HGS-TR2J MAb, CS-1008 (TRA-8) MAb, AMG 655 MAb, Apomab MAb, and LBY135 MAb.

In some embodiments, the antineoplastic agent is selected from the group consisting of an alkylating agent, an , a plant alkaloid, a cytotoxic antibiotic, a platinum compound, a methylhydrazine, a monoclonal antibody, a photodynamic/radiation therapy sensitizer, and a , or a combination thereof. In some embodiments, the antineoplastic agent is selected from the group consisting of , , , , , , , , , , , , , , , , , Streptozocin, , , Ranimustin, , , , , , , , , , , , , , , , , , , , , , , , , , , , , Paclitaxel poliglumex, , , Doxorubicin, , , , , , , , , , , , , Mitomycin, , Cisplatin, , , , Polyplatillen, , Edrecolomab, Rituximab, Trastuzumab, Alemtuzumab, Gemtuzumab, Cetuximab, Bevacizumab, Panitumumab, Catumaxomab, Ofatumumab, , , , , , Imatinib, Gefitinib, Erlotinib, Sunitinib, Sorafenib, Dasatinib, Lapatinib, Nilotinib, Temsirolimus, Everolimus, Pazopanib, Vandetanib, Afatinib, Masitinib, Toceranib, , , , , , , Miltefosine, , Estramustine, , , , Tiazofurine, , , , , , , Denileukin diftitox, , , , , Sitimagene ceradenovec, , , Omacetaxine mepesuccinate, , , and Tamoxifen.

Other objects and features will be in part apparent and in part pointed out hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1 is a pair of images depicting genomic analysis of 8p deletion in invasive cervical cancer. FIG. 1A shows identification of 8p12-21 .3 minimal deletion using 250K Nspl SNP array. Each vertical column represents a sample with genomic regions representing from pter (top) to qter (bottom) on chromosome 8 (G-band ideogram). Prefix

"T" indicates primary tumor; "CL" indicates cell line. The blue-red scale bar (-1 to +1) at the bottom represents the copy number relative to mean across the samples. Blue indicates a loss and red indicates a gain. All tumors that exhibited chromosome 8 losses are shown from largest to smallest region of deletion. Inferred copy number view of T-194 showing 8p minimal deletion from normal (2N) (red line) is shown on right. FIG. 1 B shows gene expression profile using U133A array and supervised analysis of TNFRSF10 genes and NKX3-1 mapped to a minimal deleted region (MDR) in cervical cancer lines and primary tumors. In the matrix, each row represents the gene expression relative to group mean and each column represents a sample. The scale bar (-2 to +2) on the bottom represents the level of expression with intensities of blue representing decrease and red representing increase in expression.

FIG. 2 is a series of box plots and gel images showing analysis of gene expression of decoy receptors in cervical cancer cell lines by RT-PCR. FIG. 2A and FIG. 2C are box plots showing semi-quantitative analysis of transcript levels. Promoter methylation correlated with the down regulated expression of TNFRSF10C and TNFRSF10D. ANOVA, unmethylated tumor (UM) vs. methylated tumor (M) is significant; plots show median, 25th and 75th percentile, minimum and maximum values. FIG. 2B and FIG. 2D show effect of drug treatment using inhibitors of DNA methyltransferases and HDAC on gene reactivation. Bracketed M and UM indicates methylated and unmethylated tumors, respectively. ACTB, beta actin; U, untreated; A , azacytidine; T, TSA; A T, azacytidine and TSA. Reactivation of expression of TNFRSF10D in ummethylated cell lines is indicative of involvement of other epigenetic/chromatin modifications. FIG. 3 is a series of bar graphs showing apoptosis analysis by flow cytometry in cervical cancer cell lines after exposure to TRAIL-combination drugs to assess apoptosis in relation to decoy receptor status and 8p deletion.

FIG. 4 is a series of bar graphs showing promoter methylation and gene expression of decoy receptor genes in hematologic malignancies. FIG. 4A shows frequency of promoter methylation in various lineages. MDS, myelodysplasia syndrome; AML, acute myeloid leukemia; CML, chronic myelogenous leukemia; B-ALL, Bcell acute lymphoblastic leukemia; RL/FH, reactive lymphoid/follicular hyperplasia; BL, Burkitt lymphoma; FL, follicular lymphoma; DLBCL, diffuse large B-cell lymphoma; MCL, mantle cell lymphoma; MZL, marginal zone lymphoma; MM, multiple myeloma; HD, Hodgkin disease; T-ALL, T-cell acute lymphoblastic leukemia; TCL, T-cell lymphoma. FIGS. 4B-C show analysis of gene expression of decoy receptors in promoter methylated T-ALL cell lines by RT-PCR and effect of treatment using inhibitors of DNA methyltransferases and HDAC on gene reactivation. FIG. 4B shows TNFRSF10C. FIG. 4C shows TNFRSF10D. Promoter methylation correlated with the down regulated expression of TNFRSF10C in both cell lines ( FIG. 4B) and TNFRSF10D is down-regulated in Jurkat, but detectable levels of expression seen in Karpas-45 ( FIG. 4C). ACTB, beta actin; Control, untreated; Aza, azacytidine; TSA, trichostatin A ; Aza+TSA, azacytidine and TSA.

FIG. 5 is a series of bar graphs showing apoptosis analysis by flow cytometry in T- ALL cell lines [N=14] to TRAIL combination drugs to assess cell death in relation to decoy receptor expression. TRAIL, 0.5 pg/ml; Asparaginase, 2 lU/ml; Dexamethasone, 100 nM/ml; Methotrexate, 50 nM/ml. Panel A , TNFRSF10C; Panel B, TNFRSF10D; -ve, no detectable expression; +ve, Expressed. *, Significant (P=0.05-0. 001); ** highly significant (0.001 and below).

FIG. 6 is a bar graph showing cytotoxicity analysis by MTT assay in B-cell lymphoma cell lines [N=10] after exposure to TRAIL-doxorubicin combination treatment in relation to TNFRSF10C gene expression. Dox, Doxorubicin; TRAIL, 0.5 pg/ml; Doxorubicin, 15 ng/ml. B-cell lymphoma cell lines with decreased expression of TNFRSF10C show synergistic affect by > 10-fold higher sensitivity to TRAIL -doxorubicin treatment compared to TRAIL alone.

FIG. 7 is a series of bar graphs showing cytotoxicity analysis by MTT assay in breast cancer cell lines (N=3) after exposure to TRAIL-combination drugs to assess cell survival in relation to TNFRSF10C methylation. Tarn, Tamoxifen; Dox, Doxorubicin; TRAIL, 0.5 pg/ml; Doxorubicin, 250 ng/ml; Tamoxifen, 15 mM/ml. M, methylated; UM, unmethylated. Breast cancer cell lines harboring TNFRSF10C methylation (Mx-1 and MDA-MB-231) were highly sensitive to TRAIL combined with either Tamoxifen or Doxorubicin compared to unmethylated breast cancer cell line (MCF7).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery that cervical cancer cell lines carrying an 8p deletion and decoy receptor methylation/down-regulated expression result in a high apoptotic response to TRAIL-induced combination therapy.

High-throughput genomic studies resulted in the identification of MDR at 8p12-21 .3 region, which contains a cluster of four tumor necrosis factor related super family (TNFRSF) of death receptors (DRs) (two death receptors, DR1/TNFRSF10A and DR2/TNFRSF10B; two decoy receptors, DcR1/TNFRSF10C and DcR2/TNFRSF10D). DR1, DR2, DcR1 , and DcR2 receptors express on the cell surface and play a role in extrinsic apoptotic pathway. The DR4 and DR5 genes induce apoptosis by binding to TRAIL/Apo2 ligand through their death domains and decoy receptors DcR1 and DcR2 lacking functional intracellular death domains inhibit TRAIL induced apoptosis. As reported herein, a high frequency of promoter hypermethylation and down-regulated expression of DcR1 and DcR2 decoy receptor genes occurs in cervical cancer cell lines.

Furthermore, it has been discovered that cell lines carrying genomic 8p deletion accompanied by decoy receptor methylation exhibit enhanced TRAIL-induced apoptosis compared to cell lines lacking these changes. As such, epigenetic inactivation of decoy receptor genes (TNFRSF10C/DcR1 and TNFRSF1 0D/DcR2) can serve as a biomarker for TRAIL response in cervical cancer. Identification of an 8p deletion or decoy receptor promoter hypermethylation can facilitate the correlation of cervical cancer progression with therapeutic outcome, an improved evaluation of treatment, and approaches for early detection.

Because a large proportion of B-NHL patients exhibit resistance to the existing treatments, there is a need for developing new therapies and biomarker of response, as described herein, to stratify patients that benefit from specific treatments, such as TRAIL combination drugs.

It is presently thought that 8p deletion and decoy receptor inactivation may play a role in sensitizing breast cancer cells to TRAIL combination drug therapy. Therefore, these suboptimal outcomes of conventional treatments in breast cancer patients warrants an urgent need for the development of new therapies and identification of biomarkers, as described herein, to stratify patients and predict response to a specific drug therapy.

BlOMARKER

Provided herein is a method for determining susceptibility of a cancer of a subject to TRAIL-induced apoptosis through, for example, TRAIL-combination therapy. Also provided herein is a method for stratification of subjects for TRAIL-combination therapy.

As shown above, there can be a relationship between 8p21 .3 deletion and methylation mediated inactivation of DcRs that can predict response to TRAIL-mediated apoptosis. The 8p deletion can predict adverse overall survival in cervical cancer. The presence of DcR methylation-mediated inactivation in HPV positive cell lines can be an indicator of higher rate of apoptosis to TRAIL-combination therapy.

An 8p chromosomal deletion can predict an adverse overall survival in cervical cancer. An 8p chromosomal deletion together with promoter methylation-mediated inactivation of a decoy receptor gene (e.g., TNFRSF10D and TNFRSF10C) can be an indicator of increased sensitivity to TRAIL induced apoptosis through, for example TRAIL combination drug treatment. An 8p deletion can be a deletion at 8p12-p21 .3. For example, an 8p deletion can include an 8.4 Mb minimal region of deletion (MRD) between 22,941-31,338 kb physical interval at 8p12-p21 .3.

Inactivation of a decoy receptor can independently predict response in the presence of an 8p deletion to TRAIL-combination treatment. For example, methylated- mediated inactivation of a decoy receptor and an 8p deletion can be a biomarker for increased TRAIL-induced apoptosis.

TRAIL decoy receptor genes epigenetically inactivated in T-cell neoplasms can be used as biomarkers of TRAIL-anti-leukemic drug combination therapy response to identify patients that benefit this treatment. For example, decoy receptor inactivation can be used as a biomarker for TRAIL-combination treatment in T-cell malignancies, such as neoplasms of immature T-cells (e.g., T-AA/LBL) and mature T-cells (e.g., PTCL) as occurring in, for example, cervical cancer. Use of such biomarker can provide rationale for selective use of combination agents that activate the TRAIL pathway or provide prognostic value in prediction of TRAIL response in cervical cancer or T-cell malignancies.

TRAIL decoy receptor genes epigenetically inactivated in B-cell lymphomas can be used as a biomarkers of TRAIL-combination drug therapy response to identify patients that benefit this treatment. For example, TNFRSF10C inactivation can be used as a biomarkerfor TRAIL-combination drug treatment in B-cell lymphomas such as lymphomas of germinal centre origin (e.g., Burkitt's lymphoma, follicular lymphoma and diffuse large B-cell lymphoma) as occurring in, for example, cervical cancer and T-ALL. Use of such biomarker can provide rationale for selective use of combination agents that activated TRAIL pathway or provide prognostic value in prediction of TRAIL-combination drug response in mature B-cell lymphomas.

TRAIL decoy receptor genes epigenetically inactivated in breast cancer can be used as biomarkers of TRAIL-combination drug therapy response to identify patients that benefit this treatment. For example, TNFRSF10C inactivation can be used as a biomarker for TRAIL-combination drug treatment in breast cancer patients as occurring in, for example, cervical cancer, T-ALL, and B-cell Non-Hodgkin Lymphoma. Use of such biomarker can provide rationale for selective use of combination agents that activated TRAIL pathway or provide prognostic value in prediction of TRAIL-combination drug response in patients with breast cancer.

Decreased expression of a decoy receptor in the presence of an 8p deletion can predict response to TRAIL-combination treatment. For example, methylated-mediated decreased expression of a decoy receptor and an 8p deletion can be a biomarker for increased TRAIL-induced apoptosis.

A decoy receptor that is methylated or has reduced expression can be, for example, TNFRSF10C or TNFRSF10D. As another example, decoy receptor that is methylated or has reduced expression can be, for example, a methylated promoter of TNFRSF10C. As another example, decoy receptor that is methylated or has reduced expression can be, for example, a methylated promoter of TNFRSF10D. As another example, decoy receptor that is methylated or has reduced expression can be, for example, a methylated promoter of TNFRSF10C and TNFRSF10D. It is thought that suppression of DcR1 and DcR2 in resistant cells that express these genes normally but harboring 8p deletion can result in sensitivity to TRAIL. On the other hand, restoration of DcR1 and DcR2 function in sensitive cells harboring 8p deletion and down regulated expression of DcRs can acquire resistance. Thus, 8p deletion in association with DcR inactivation can be used as a biomarker for TRAIL combination therapy. DETECTION

One aspect provides a diagnostic assay to detect one or more of an 8p deletion, decoy receptor inactivation, or reduced decoy receptor expression in a subject. Each of 8p deletion, decoy receptor inactivation, or reduced decoy receptor expression can be as described above. For example, the diagnostic assay can detect two or more of an 8p deletion, decoy receptor promoter hypermethylation, or lack of or reduced decoy receptor expression. As another example, the diagnostic assay can detect all of an 8p deletion, decoy receptor methylation, or lack of or reduced decoy receptor expression. Such a test can be used for clinical specimens of a subject to stratify or assign invasive cervical cancer cases for TRAIL-combination therapy.

Detection of an 8p deletion can be according to Fluorescence In Situ Hybridization (FISH). This and other means of detecting an 8p deletion is within the skill of the art.

Detection of decoy receptor methylation can be according to methylation specific PCR (MSP). For example, detection of promoter methylation of TNFRSF10C or TNFRSF10D can be according to MSP. This and other means of detecting decoy receptor methylation (e.g., TNFRSF10C or TNFRSFIOD methylation) is within the skill of the art.

Detection of decoy receptor expression levels can be according to a semi quantitative method of estimation of protein. For example, detection of decoy receptor expression levels can be according an immunohistochemistry assay. As another example, detection of decoy receptor expression levels can be according an immunofluorescence assay. A semi-quantitative method of estimating decoy receptor protein (e.g., TNFRSF10C or TNFRSFIOD protein) is within the skill of the art.

A diagnostic assay can also include identification of HPV status and type. For example, HPV typing can be according to conventional protocols in cancer (e.g., cervical cancer) management.

A diagnostic assay including one, two, three or more of the detection protocols described herein can provide a robust biomarker-directed test to detect 8p deletion, decoy receptor promoter hypermethylation, or decoy receptor reduced expression that can be applicable in, for example, clinical specimens. TRAIL COMBINATION THERAPY

Provided herein are diagnostic assays that can determine sensitivity of a cancer to TRAIL-induced apoptosis, especially through TRAIL combinatorial therapy. TRAIL combinatorial therapy can include administration of one or more TRAIL agonists and one or more antineoplastic agents. An antineoplastic agent can be administered at non-toxic dose in combination with TRAIL therapy so as to increase TRAIL-induced-apoptosis in cancer cells.

TRAIL therapy can be as described in Bellail et al. 2009 Reviews on Recent Clinical Trials 4, 34-41 , incorporated herein by reference. For example, TRAIL apoptotic pathway can be targeted by recombinant human TRAIL (rhTRAIL) ligands or its agonistic antibodies against DR4 and DR5. Such approaches are presently in clinical trials. As another example, TRAIL therapy can comprise administering a TRAIL agonist selected from Apo2L/TRAIL (Genentech, Amgen), HGS-ETR1 MAb (Human Genome Sciences), HGS-ETR2 MAb (Human Genome Sciences), HGS-TR2J MAb (Human Genome Sciences), CS-1008 (TRA-8) MAb (Daiichi Sankyo), AMG 655 MAb (Amgen), Apomab MAb (Genentech), or LBY135 MAb (Novartis).

As described herein, presence of an 8p deletion or methylation of a decoy receptor promoter can indicate susceptibility to a TRAIL combinatorial therapy. TRAIL therapy can be administered in combination with one or more antineoplastic agent. For example, TRAIL therapy can be administered in combination with one or more chemotherapeutic agents.

An antineoplastic agent can be a chemotherapeutic agent. An antineoplastic agent can be an alkylating agent, an anti-metabolite agent, a plant alkoloid or terpenoid, a vinca alkaloid, a podophyllotoxin, a , or a .

Exemplary antineoplastic agents include, but are not limited to, alkylating agents (e.g., analogues, alkyl sulfonates, ethylene imines, , epoxides, or other alkylating agents), (e.g., folic acid analogues, purine analogues, pyrimidine analogues), plant alkaloids and other natural products (e.g., vinca alkaloids and analogues, podophyllotoxin derivatives, colchicine derivatives, , or other plant alkaloids and natural products), cytotoxic antibiotics and related substances (e.g., actinomycines, and related substances, or other cytotoxic antibiotics), platinum compounds, methylhydrazines, monoclonal antibodies, sensitizers used in photodynamic/radiation therapy, protein kinase inhibitors, or combinations thereof. An antineoplastic agent can be a nitrogen mustard analogue selected from Cyclophosphamide, Chlorambucil, Melphalan, Chlormethine, Ifosfamide, Trofosfamide, Prednimustine, or Bendamustine. An antineoplastic agent can be an alkyl sulfonate selected from Busulfan, Treosulfan, or Mannosulfan. An antineoplastic agent can be an ethylene imine selected from Thiotepa, Triaziquone, or Carboquone. An antineoplastic agent can be a selected from Carmustine, Lomustine, Semustine, Streptozocin, Fotemustine, Nimustine,or . An antineoplastic agent can be a folic acid analogue selected from Methotrexate, Raltitrexed, Pemetrexed, or Pralatrexate. An antineoplastic agent can be a selected from Mercaptopurine, Tioguanine, Cladribine, Fludarabine, Clofarabine, or Nelarabine. An antineoplastic agent can be a selected from Cytarabine, Fluorouracil, Tegafur, Carmofur, Gemcitabine, Capecitabine, Azacitidine, Decitabine, Fluorouracil combinations, or Tegafur combinations. An antineoplastic agent can be a vinca alkoloid or analogue selected from Vinblastine, Vincristine, Vindesine, Vinorelbine, or Vinflunine. An antineoplastic agent can be a podophyllotoxin derivative selected from Etoposide or Teniposide. An antineoplastic agent can be a colchicine derivative selected from Demecolcine. An antineoplastic agent can be a taxane selected from Paclitaxel, Docetaxel, or Paclitaxel poliglumex. An antineoplastic agent can be a natural product selected from Trabectedin. An antineoplastic agent can be an actinomycines selected from Dactinomycin. An antineoplastic agent can be an anthracycline selected from Doxorubicin, Daunorubicin, Epirubicin, Aclarubicin, Zorubicin, Idarubicin, Mitoxantrone, Pirarubicin, Valrubicin, Amrubicin, or Pixantrone. An antineoplastic agent can be a cytotoxic antibiotic selected from Bleomycin, Plicamycin, Mitomycin, or Ixabepilone. An antineoplastic agent can be a platinum compound selected from Cisplatin, Carboplatin, Oxaliplatin, Satraplatin, or Polyplatillen. An antineoplastic agent can be a methylhydrazine selected from Procarbazine. An antineoplastic agent can be a monoclonal antibody selected from Edrecolomab, Rituximab, Trastuzumab, Alemtuzumab, Gemtuzumab, Cetuximab, Bevacizumab, Panitumumab, Catumaxomab, or Ofatumumab. An antineoplastic agent can be a sensitizer selected from Porfimer sodium, Methyl aminolevulinate, Aminolevulinic acid, Temoporfin, or Efaproxiral. An antineoplastic agent can be a protein kinase inhibitor selected from Imatinib, Gefitinib, Erlotinib, Sunitinib, Sorafenib, Dasatinib, Lapatinib, Nilotinib, Temsirolimus, Everolimus, Pazopanib, Vandetanib, Afatinib, Masitinib, or Toceranib. An antineoplastic agent can be selected from Amsacrine, Asparaginase, Altretamine, Hydroxycarbamide, Lonidamine, Pentostatin, Miltefosine, Masoprocol, Estramustine, Tretinoin, Mitoguazone, Topotecan, Tiazofurine, Irinotecan, Alitretinoin, Mitotane, Pegaspargase, Bexarotene, Arsenic trioxide, Denileukin diftitox, Bortezomib, Celecoxib, Anagrelide, Oblimersen, Sitimagene ceradenovec, Vorinostat, Romidepsin, Omacetaxine mepesuccinate, Eribulin, or Camptothecin.

An antineoplastic agent can include cisplatin. An antineoplastic agent can comprise a combination of hycamtin and cisplatin. A combination of hycamtin and cisplatin is FDA approved for treatment of late-stage (IVB) cervical cancer.

One of ordinary skill will understand this listing of antineoplastic agents is exemplary and any anticancer agent can be used in combination with a TRAIL agonist where such combination results in increased apoptosis compared to either agent alone.

As described herein, cells having an 8p deletion, methylated promoter of decoy receptors, or reduced decoy receptor expression can exhibit increased apoptosis in response to combinatorial therapy comprising administration of a TRAIL agonist and an antineoplastic agent. Administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL-induced apoptosis as compared to administration of either alone. Administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL- induced apoptosis in excess of an additive effect of administration of either alone. Administration of a TRAIL agonist and an antineoplastic agent can synergistically increase TRAIL-induced apoptosis as compared to individual administration.

For example, administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL-induced apoptosis by at least about 10% as compared to administration of either alone. As another example, administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL-induced apoptosis by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, or more, as compared to administration of either alone.

MOLECULAR ENGINEERING

Design, generation, and testing of the variant nucleotides, and their encoded polypeptides, having the above required percent identities and retaining a required activity of the expressed protein is within the skill of the art. For example, directed evolution and rapid isolation of mutants can be according to methods described in references including, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991 ) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art could generate a large number of nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art. Generally, conservative substitutions can be made at any position so long as the required activity is retained.

Nucleotide and/or amino acid sequence identity percent (%) is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A to, with, or against a given sequence B (which can alternatively be phrased as a given sequence A that has or comprises a certain percent sequence identity to, with, or against a given sequence B) can be calculated as: percent sequence identity = X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A .

"Highly stringent hybridization conditions" are defined as hybridization at 65 °C in a

6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (Tm) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize. In general, the melting temperature for any hybridized DNA:DNA sequence

+ can be determined using the following formula: Tm = 8 1.5 °C + 16.6(logi 0[Na ]) + 0.41 (fraction G/C content) - 0.63(% formamide) - (600/I). Furthermore, the Tm of a DNA:DNA hybrid is decreased by 1-1 .5°C for every 1% decrease in nucleotide identity (see e.g., Sambrook and Russel, 2006).

Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717;

Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. The transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.

Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Methods of down-regulation or silencing genes are known in the art. For example, expressed protein activity can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerhead ribozymes and small hairpin RNA; Helene, C , et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, describing RNAi). RNAi molecules are commercially available from a variety of sources {e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.

FORMULATION

The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.

Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.

THERAPEUTIC METHODS

Also provided is a method of treating a cervical cancer in a subject in need administration of a therapeutically effective amount of a TRAIL agonist and an antineoplastic agent, so as to increase TRAIL-mediated apoptosis.

Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing cervical cancer. A subject in need of the therapeutic methods described herein can be a subject having or diagnosed as having one or more of an 8p chromosomal deletion, a methylation- associated inactivation of a decoy receptor, or reduced decoy receptor expression levels in a tumor of the subject. A determination of the need for treatment can be according to diagnostic assays described herein. A determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, preferably a mammal, more preferably horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, guinea pigs, and chickens, and most preferably a human.

An effective amount of a TRAIL agonist and an antineoplastic agent described herein is generally that which can induce apoptosis in cancer cells or a tumor of the subject.

According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. When used in the treatments described herein, a therapeutically effective amount of a TRAIL agonist and an antineoplastic agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to induce apoptosis in cancer cells or a tumor of the subject. A TRAIL agonist and an antineoplastic agent can be administered in the same composition. A TRAIL agonist and an antineoplastic agent can be administered in different compositions.

The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio

LD50/ED5 0, where large therapeutic indices are preferred.

The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical , 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071 375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

Administration of a TRAIL agonist and an antineoplastic agent can occur as a single event or over a time course of treatment. For example, a TRAIL agonist and an antineoplastic agent can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for cervical cancer, T-cell hematologic malignancies, B-cell NHL, or breast cancer.

A TRAIL agonist or an antineoplastic agent can be administered simultaneously or sequentially. A TRAIL agonist or an antineoplastic agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an antiinflammatory, or another agent. For example, a TRAIL agonist or an antineoplastic agent can be administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a TRAIL agonist or an antineoplastic agent, an antibiotic, an antiinflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a TRAIL agonist, an antineoplastic agent, an antibiotic, an antiinflammatory, or another agent. A TRAIL agonist or an antineoplastic agent can be administered sequentially with an antibiotic, an antiinflammatory, or another agent. For example, a TRAIL agonist or an antineoplastic agent can be administered before or after administration of an antibiotic, an antiinflammatory, or another agent. ADMINISTRATION

Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g. , systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 µ η ), nanospheres (e.g., less than 1 µ η ), microspheres (e.g., 1- 100 µ η ), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition is administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier- based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.

SCREENING

Also provided are methods for screening of agents that in combination with a TRAIL agonist provide for increased TRAIL-induced apoptosis as compared to the TRAIL agonist or antineoplastic agent alone. Also provided are methods for screening of TRAIL agonists that in combination with an antineoplastic agent provide for increased TRAIL- induced apoptosis as compared to the antineoplastic agent or TRAIL agonist alone.

The subject methods find use in the screening of a variety of different candidate molecules (e.g., potentially therapeutic candidate molecules). Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.

Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups. The candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. A candidate molecule can be a compound in a library database of compounds. One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-1 82). One of skill in the art will also be familiar with a variety of search engines to identify commercial sources or desirable compounds and classes of compounds for further testing (see e.g., ZINC database; eMolecules.com; and electronic libraries of commercial compounds provided by vendors, for example: ChemBridge, Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicals etc).

Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds. A lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943-

3948). In contrast, a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249). Preferably, initial screening is performed with lead-like compounds.

When designing a lead from spatial orientation data, it can be useful to understand that certain molecular structures are characterized as being "drug-like". Such characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia. While it is not required for drugs to meet all, or even any, of these characterizations, it is far more likely for a drug candidate to meet with clinical successful if it is drug-like.

Several of these "drug-like" characteristics have been summarized into the four rules of Lipinski (generally known as the "rules of fives" because of the prevalence of the number 5 among them). While these rules generally relate to oral absorption and are used to predict bioavailability of compound during lead optimization, they can serve as effective guidelines for constructing a lead molecule during rational drug design efforts such as may be accomplished by using the methods of the present disclosure. The four "rules of five" state that a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms). Also, drug-like molecules typically have a span (breadth) of between about 8A to about 15A.

KITS

Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to reagents for detection assays described herein, a TRAIL agonist, or an antineoplastic agent. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.

In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

EXAMPLE 1

Tumor cell lines and tissue culture. Cells lines for Example 8 include nine CC cell lines (C-4I, C-33A, HT-3, Ca Ski, ME-180, MS751, SW756, HeLa, and SiHa) from ATCC and 3 other CC cell lines (MARQ, Goerke F12, and MRI-H-186). Cell lines for Example 6 include nine T-ALL (CUTTL1, HPB-ALL1, JURKAT, Karpas-45, MOLT-4, MOLT-15,

MOLT-16, P12-lchikawa, T-ALL1), two B-ALL (REH, RS4;1 1), and six PTCL (MT-1, MT-2, HUT-102, C5MJ, TL-Om1, and ED40515). Cell lines for Example 11 include ten B-cell NHL cell lines (Daudi, Raji, SUDHL-4, SU-DHL-4, SU-DHL-5, SU-DHL-8, SU-DHL-10, LY-

1, LY-3, WSU, and Farage) and three breast cancer cell lines (MCF7, Mx-1, and MDA- MB-231). These cell lines have been characterized by molecular cytogenetic methods, and used for generating data on TRAIL-induced apoptosis presented here [31].

For Example 6, cells grown in culture are treated with defined drug concentrations and periods (e.g. 5-Aza-2'deoxycytidine, 5 µΜ; trichostatin, 200 nM; rhTRAIL, 0.5-1 pg; dexamethasone, 50-150 nM; doxorubicin, 10-25 ng). For Example 8, cells grown in culture are treated with defined drug concentrations and periods (5-Aza-2'deoxycytidine, 5 µΜ; trichostatin, 200 nM; rhTRAIL, 0.5-1 pg; actinomycin D, 0.5-1 g; cisplatin, 1-5 pg; doxorubicin, 100-200 nM; and tamoxifen, 100-200 nM). The agonistic antibodies include for TRAILRI (mapatumumab) and TRAILR2 (lexatumumab) (Human Genome Sciences). Cells in culture are exposed to gamma radiation using Gammacell 40 Cesium Unit.

Fluorescence in situ hybridization (FISH): FISH is according to conventional methods on metaphases prepared from stimulated blood cultures, cell lines, cytological smears and paraffin/frozen-sections. BAC DNA is prepared and labeled by nick- translation using spectrum orange or spectrum green fluorochromes by standard methods. Centromere-specific probes are obtained from Abbott Molecular (Downers Gove, IL). Normal variation of the FISH signal pattern is established for each tissue type (frozen, paraffin, smear, metaphase) on 5 controls and the signals are scored by 3 investigators (including a pathologist). Normal cut-off values above which specimen would be called positive for deletion are established at 95% confidence level using beta inverse function.

MSP, Sequenom Epityper, bisulphite sequencing and RT-PCR: MSP is performed using the standard methods by converting genomic DNA using EpiTect bisulphite kit (Qiagen, CA) with appropriate primer sets. Quantitative methylation analysis will be performed using Sequenom MALDI-TOI mass spectrometry platform as per manufacturer specifications (Sequenom, San Diego, CA). Briefly, 50 ng of bisulphite converted genomic DNA is PCR amplified using T7-promoter tagged primers spanning the CpG island. Using the manufacturer's protocols, the samples are analyzed with the Sequenom MALDI-TOF MS Compact Unit and the Sequenom kit. The quantitative fragment mass data generated by the MALDI-FOF MS and EpiTYPER software is further subjected to quality control analysis on duplicate reactions for each primer set. The final data output consists of average methylation measurements of each informative CpG of duplicate experiments. The software estimates the relative methylation status by the sum of the intensities of the methylated and unmethylated components. After initial quality control, the data is further filtered to exclude the cases that yielded less than 80% for all informative CpG units within an amplicon/sample pair. The methylation data generated after QC is subjected to one- dimensional hierarchical clustering analysis to identify relative methylation of the test sample compared to controls. Bisulphite sequencing of MSP products after cloning into pCR2.1 TOPO vector (Invitrogen) is by standard PCR methods [32]. Semi-quantitative PCR or real-time PCR is performed on reversed transcribed RNA isolated from cell lines using Applied Biosystems 7500 PCR system and by standard methods (Foster City, CA).

siRNA, shRNA, lentiviral, and plasmids transfections. For in vitro experiments, oligonucleotides corresponding to the sequences of each gene are commercially obtained for siRNA experiments along with negative controls. Transfections are carried out, serially or simultaneously if two genes targeted, using reagents such as Oligofectamine (Invitrogen). Efficiency of transfection is judged by either RT-PCR or by Western analysis on cells collected after 48-72 hr after transfection. Second transfection is done if the reduction of RNA/protein is incomplete in the first transfection. Full-length ORF is cloned in pcDNA and transfected along with vector controls by standard methods and stably selected clones are used in both in vitro and in vivo testing of TRAIL-combination treatments. For stable transfections, predesigned inducible HuSH 29mer shRNA constructs against the in pGFP-V-RS expression vectors and/or lentiviral vectors along with the controls are obtained from commercial sources. Alternatively, lentiviral constructs are generated using appropriate vectors. TetR-expression cell lines are generated or selected for stable cell line. Lentiviral production and infections is performed using standard methods and titers are monitored using GFP visualization [33]. Stable cell lines generated by these transfections and transductions are used for TRAIL combination treatment experiments in vitro and in vivo.

Tissue, Microarray and Immunohistochemical Analysis: Tissue microarrays of formalin-fixed, paraffin embedded tissue specimens were constructed on 75 invasive cervical cancers. Immunohistochemistry is performed on paraffin tissue section after antigen retrieval and antigen detection using appropriate antibodies by standard methods using Ventana staining platform. If appropriate, densitometric measurements of color intensity is performed on CAS2000 image analysis system. Immunofluoresence according to conventional protocols is an alternative approach to examine the specificity compared to IHC.

Cytotoxicity and flow cytometry: Cell viability and cytotoxicity against TRAIL and combination of other drugs is assessed by standard colorimetric MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Invitrogen, Carlsbad, CA). The absorbance is measured at 490 nm with a microtiter plate reader. All treatments are performed in four replicate wells and in 3 repeats. Apoptosis is measured using Pacific Blue™ Annexin V/SYTOX® AADvanced™ Apoptosis Kit (Invitrogen, Carlsbad), which detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to violetfluorescent Pacific Blue dye and dead cell using SYTOX

AADvanced stain. LSR II flow cytometry instrument (BD Biosciences, San Jose) is used. All experiments are done in duplicate for each drug combination.

Western blot analysis: Western blot analysis is performed by standard methods and detection by chemiluminescence reagent. Immunoblotting is performed using relevant antibodies including TRAIL R1, TRAIL R2, TRAIL R3, TRAIL R4, c-FLIP, caspase 8, caspase 9, p53, HPV E6 and E7.

Tumor xenograft and evaluation of drug response: Cell lines are genetically modified to express ffluc gene by transfecting the vector containing the luciferase (Promega). Exponentially growing cells are collected and embedded in Matrigel (BD Biosciences) at 1X108 cells per ml. 10 million cells are injected into dorsal flank using 1-cc TB syringe in athymic nu/nu mice (if needed NOD-SCID mice will also be tested). The IC50 value for each drug is determined for each cell line. For unmodified cell lines, in group one 10 mice are injected for each cell line and a control group of 5 with vehicle injection. For the modified cell lines with 'knock-down' or 'knock-in' of specific DcR genes, 10 mice each are injected for modified and parental cell line. Tumor growth is monitored daily to allow 0.2-0.5 cm2 on the longest axis before initiating the drug treatment. Alternatively, in vivo tumor growth is monitored once a week by the luciferase activity by using bioluminescent image on anesthetized animals. The imaging is performed at Confocal and Specialized Microscopy. Assessment of in vivo efficacy of TRAIL combination therapy of xenografts is performed by tumor-derived biolumenesence using

CCD camera ( IVIS Spectrum System, Xenogen, Caliper Lifesciences) after injecting D- luciferin. Photons from the bioluminescence signal are measured as total photon flux normalized to exposure time and surface areas and expressed in units of photon/second/cm 2/steradian. Animals are monitored daily for normal behavior, weight loss, and other signs of clinical end points. The anti-tumor effect of drug therapy is evaluated according to the sizes of tumors for 4 weeks and latter. Histological and cytotoxicity studies, when needed, are performed on normal tissues and excised tumors.

Statistical analysis: Quantitative comparisons will be performed by Student 't' test, the chi-square and Fisher exact tests. Statistical analyses is performed using the GraphPad Prism software (LaJolla, CA). EXAMPLE 2

This example shows a complex pattern of genomic alterations at 8p21 .3 including large deletions, methylation of DcR genes and their associated down-regulated expression, and reactivation of DcR expression upon inhibiting DNA methyltransferases.

High-throughput genomic techniques resulted in the identification of an 8.4 Mb minimal region of deletion (MRD) between 22,941-31,338 kb physical interval at 8p12- p21 .3 in 38% of 80 CC analyzed by Affymetrix SNP arrays. Further studies demonstrated that patients carrying 8p deletion were at a 2-fold higher risk of death. The 8p12-21 .3 deletion was thus thought to contain critical tumor causing genes in cervical cancer.

To identify down-regulated genes associated with 8p deletion in CC, gene expression analysis was performed on 92 probe sets comprising of 53 known genes mapped to MRD obtained from U133A arrays analyzed on 33 primary tumors, 9 cell lines, and 20 normal cervical epithelia. Utilizing the criteria of 1.75-fold change at 90% confidence interval between the group means of normal and tumor, loss of expression in NKX3-1 gene was found in over 90% tumors (see e.g., FIG. 1B) (28). But no correlation was found between NKX3-1 expression levels and 8p genomic deletion and no mutations/promoter methylation modifications were found in the remaining allele, suggesting that NKX3-1 silenced state is not directly linked to 8p MRD. These data, therefore, suggested potential involvement of other critical tumor suppressor genes at 8p deletion in CC transformation or progression.

Next examined was the transcript levels of 3 (TNFRSF10B, TNFRSF10C and TNFRSF1 0D) of the 4 TNFRSF1 0 gene cluster mapped to 8p MRD that were present on U133A array. TNFRSF10D down-regulation was found in 69% of invasive CC (see e.g., FIG. 1B), while the pattern of expression of the other decoy receptor gene TNFRSF1 0C was not consistently down-regulated and the proapoptotic death receptor TNFRSF10B showed higher levels of expression in tumors compared to normal (see e.g., FIG 1B). TNFRSF family of decoy receptor genes is known to be epigenetically inactivated in multiple human tumors.

To address the role of TRAIL receptors and their relation to 8p deletion in CC, the receptor genes for promoter hypermethylation was examined. We found no evidence of methylation in the DR genes, while the decoy receptor TNFRSF1 0C showed methylation in 45.5% and TNFRSF10D in 5.7% of CC. Because the decoy receptor expression using U133A array was unclear in relation to 8p deletion, the expression studies were further extended by RT-PCR to correlate with the methylation status. Results of these studies showed that epigenetic alterations play a role in the repressed state of DcR genes and reactivation occur after inhibiting methyltransferases and histone deacetylases (see e.g., FIG. 2). Thus, was shown that DcRs are the target of epigenetic silencing at 8p MRD and are an alteration in CC tumorigenesis.

EXAMPLE 3

Conventional treatment of CC employs cisplatin followed by radiotherapy. Cisplatin treatment results in DNA damage by intercalating, which activates apoptotic pathway when p53 mediated DNA damage repair fails and also acts as a radio-sensitizer. But the response rates of cisplatin as a single agent are low in recurrent and metastatic CC and 5-year survival rates remain low. Thus, it is important to develop effective therapeutic interventions and the host genomic alterations may play critical roles in treatment response.

Because the TNFRSF genes involved in 8p deletion and inactivation of DcRs likely promote apoptosis, these alterations were thought to play a role in TRAIL response. Various cervical cancer cell lines with 8p deletions and TNFRSF10C or TNFRSFIOD inactivation were exposed to TRAIL alone and in combination with cisplatin, actinomycin D, or radiation to assess cytotoxicity and apoptosis.

Results showed that treatment of TRAIL, cisplatin, actinomycin D and ionizing radiation alone showed slightly reduced cellular viability and increased apoptosis in cell lines carrying 8p deletion and methylated/decreased expression of TNFRSF10D or

TNFRSF10C compared to the cell lines without these changes (Fig. ). However, very high and synergistic affect in cytotoxicity and apoptosis was seen in cell lines with TNFRSF10D methylated with decreased expression in combination of TRAIL with cisplatin or actinomycin D or radiation. Although less strikingly, a similar affect was observed in cell lines exhibiting TNFRSF10C down regulation associated with promoter hypermethylation (Fig. C). Therefore, in these in vitro experiments, we demonstrated that the tumor cells that carry a combination of methylation and down-regulated transcription of one or both decoy receptors in the presence of 8p deletion elicit efficient antitumor effects to TRAIL-combination treatment. Based on this data, we speculate that CC patients exhibiting 8p deletion and decoy receptor inactivation benefit from TRAIL combination therapy and may serve as biomarkers for treatment response. EXAMPLE 4

Because TNFRSF family of decoy receptors are known to be epigenetically inactivated by promoter hypermethylation in multiple human tumors [16] but their status in TCL is unknown, a wide-variety of hematologic malignancies were examined. Methods are according to Example 1 unless otherwise described.

It was found that the promoter hypermethylation is restricted to lymphoid-lineage malignancies (See e.g., FIG. 4A). Specifically, 45% of T-cell malignancies showed hypermethylation of one or both decoy receptors suggesting that inactivation of these genes is a frequent event in TCL. In addition, by testing 9 T-ALL cell lines, it was found DcR1 methylation in all cell lines, while 8 of 9 cell lines had DcR2 methylation (see TABLE 1). RT-PCR analysis showed decreased/down-regulated expression of DcR1 in all 9 cell lines and reactivation after inhibiting methyltransferases and histone deacetylases (see e.g., FIG. 4B). A complete lack of transcription of DcR2 was found in 5 of 8 methylated cell lines. The remaining 3 cell lines (Karpas-45, MOLT-15 and T-ALL1) showed detectable levels of expression (see e.g., FIG. 4C). All the 8 methylated cell lines showed evidence of reactivation after inhibiting methyltransferases. Complete lack of DcR2 expression in an unmethylated cell line MOLT-16 and failure to reactivate after azacytidine treatment suggested another mechanism of inactivation.

These data suggest that epigenetic alterations play a major role in the repressed state of DcR genes and can serve as targets of response to TRAIL treatment.

EXAMPLE 5

Response rates for T-ALL/LBL and T-cell lymphomas are low using the existing treatments of TCL using standard anti-leukemic agents (such as dexamethasone, asparaginase, methotrexate, nelarabine, bortezomib) targeting different pathways [2, 10, 17-20]. Thus, in TCL as a group, it is important to develop effective therapeutic interventions. Host genomic alterations and the associated pathways can provide targets to test treatment response [21].

Methods are according to Example 1 unless otherwise described.

Because over expression of DcRs likely inhibits apoptosis, TCL cases exhibiting decoy receptor inactivation may respond to TRAIL combination anti-leukemic drug therapies. To demonstrate this, nine T-ALL cell lines harboring TNFRSF10C and/or TNFRSF10D methylation and transcriptional inactivation were exposed to TRAIL alone and in combination with Asparaginase (2 lU/ml); Dexamethasone (100 nM/ml); or Methotrexate (50 nM/ml) to assess cytotoxicity and apoptosis.

Results showed that although use of single agents TRAIL, Asparaginase, Dexamethasone, or Methotrexatealone showed slightly increased apoptosis in T-ALL cell lines, synergistic affect was seen in combination of TRAIL with Asparaginase, Dexamethasone, or Methotrexatealone (see e.g., FIG. 5). Furthermore, cell lines carrying methylation with residual expression of TNFRSF10C or 10D were less sensitive to TRAIL-combination treatment (see e.g., FIG. 5).

Thus, the T-ALL cells that carry a combination of methylation and low levels of transcription of one or both decoy receptors can determine the anti-tumor effects to TRAIL combination treatment. Thus supports that TCL exhibiting decoy receptor inactivation can benefit from TRAIL-combination therapy.

EXAMPLE 6

Because it was found that epigenetic-mediated inactivation of decoy receptors independently predict response to TRAIL combined with dexamethasone or doxorubicin in

T-ALL cell lines, it was thought that suppression of DcR1 and DcR2 in normally expressed TRAIL-resistant cell lines could become sensitive to TRAIL and restoration of their function could result in resistance. Thus can establish decoy receptor inactivation as a biomarkerfor TRAIL-combination therapy in TCL.

Methods are according to Example 1 unless otherwise described.

All available T-ALL (N=9), B-ALL (N=2) and PTCL (N=6) cell lines are characterized for methylation by MSP and bisulphite sequencing and expression by RT- PCR or real-time RT-PCR. Established MTT and flow cytometry methods are used to identify the TRAIL and anti-leukemic drug response in relation to decoy receptor methylation/inactivation status. A number of commonly used drugs (namely dexamethasone, doxorubicin, methotrexate, L-asparaginase, nelarabine, bortezomib) clinically relevant for TCL are tested.

Such studies will confirm the relationship of decoy receptor inactivation with TRAIL response and identify several additional sensitive and resistant cell lines (Table 1). TABLE 1: Methylation and expression of decoy receptor genes in T-ALL cell lines and their response to TRAIL-chemotherapy. -, lack of expression. +, detectable expression by RT-PCR. M, methylated. UM, unmethylated. Dex, dexamethasone. Dox, doxorubicin.

Cell JURK MOLT- MOLT CUTT P12- Karpa T- MOLT HPB-

line AT 16(sensi -4 L 1 lchika s-45 ALL1 -15 ALL1 (sensit tive) (sensit (sensit wa (resist (resist (resist (resist ive) ive) ive) (sensit ant) ant) ant) ant) ive)

Dcr1 M M M M M M M M M methyl ation

DcR1 ------+ expres sion DcR2 M UM M M M M M M M methylation

DCR2 + + + expression

Cyto Percent Live Cells

toxixity assay

TRAIL 12.1 48.5 88.1 36.9 83.1 52 100 9 1.5 95.2

Dex 9 1 22 68.9 89.2 29.3 86.3 46.5 82.3 73.8

TRAIL + 9.4 15 65.8 29.6 20.8 52.9 48.1 76.4 75.6 Dex

Dox 100.1 53.3 8 1.9 94.9 97.9 76.2 40.4 50.5 113

TRAIL + 5 10.2 7.5 22.1 64.2 32.9 58.9 40.8 9 1.3 Dox

As previous attempts of TRAIL monotherapy have been ineffective in cell lines and primary tumors of T-cell origin [17, 19, 20, 23, 24], it is thought that combination therapies targeting decoy receptors are more effective.

EXAMPLE 7

Gene depletion and over-expression approaches are employed to determine the relative contribution of each of the genes and their synergistic affect in mediating apoptotic response using the following strategies.

Methods are according to Example 1 unless otherwise described.

In knockdown experiments, siRNA and shRNA approaches are employed to generate transient and stable transfectants, respectively, of one or both genes. Two unmethylated/normally expressed cell lines that exhibit resistance to TRAIL-drug induced apoptosis are chosen to transfect either TNFRSF10D or TNFRSFIOC alone or together. Initially using siRNA approach, recombinant human TRAIL (rhTRAIL) in combination with one or two of the most effectively responded drugs are tested for assessing cytotoxicity and apoptosis. Such experiments show whether inactivation of TNFRSF10D and TNFRSF10C alone is sufficient to sensitize the cells to TRAIL therapy, or inactivation of both decoy receptors have synergistic affect.

EXAMPLE 8:

This example shows gene depletion and over-expression approaches to demonstrate contribution of DcR genes and synergistic effect in mediating apoptotic response. Function of DcR genes are restored to confirm that over expression results in resistance to TRAIL treatment.

Methods are according to Example 1 unless otherwise described.

TNFRSF10D and TNFRSF10C are targeted by transfecting full length ORF clones either or both using pcDNA vectors and lentiviral ORF vectors to obtain high expression. Two cell lines that exhibit promoter methylation of TNFRSF10D and/or TNFRSF10C and down-regulated expression of respective transcripts (see TABLE 2) are chosen (e.g., MS751 and SW756, both having 8p deletion and resistance to TRAIL-cisplatin induced apoptosis).

MS751 showed no evidence of methylation or down-regulated expression in the DcRs. The SW756 cell line exhibited TNFRSF10C methylation and down-regulated expression, but TNFRSF10D is unmethylated and transcription is at the normal level. TNFRSF10D or TNFRSF10C are transfected alone or together. Initially using siRNA approach, recombinant human TRAIL (rhTRAIL) is tested in combination with cisplatin, actinomycin D or radiation for assessing cytotoxicity and apoptosis.

The readout of these experiments demonstrates (i) whether inactivation of TNFRSF10D and TNFRSF10C alone is sufficient to sensitize the cells to TRAIL therapy, (ii) inactivation of both DcRs will have synergistic affect, and (iii) 8p deletion is essential for the TRAIL response.

An alternative approach is to restore the function of DcR genes to demonstrate that over expression results in resistance to TRAIL treatment. TNFRSF10D and TNFRSF10C are targeted by transfecting full length ORF clone either or both using pcDNA vectors and/or lentiviral ORF vectors with the aim to obtain high expression of these genes. Three cell lines ME-180, C-4I, and C-33A are used. Both ME-180 and C-4I cell lines carry 8p deletion, while C-33A is disomy for 8p. All three cell lines exhibited promoter methylation and down regulated expression of TNFRSF10C. The cell lines ME-180 and C-33A also showed methylation and down regulated expression of TNFRSF10D, while C- 4 I is unmethylated and expressed at normal levels. ME180 showed highest apoptotic response to TRAIL-combination therapy and the C-33A cell line was resistant (see e.g., TABLE 2). The C-4I cell line showed moderate apoptotic response (see e.g., TABLE 2).

TABLE 2 : 8p deletion, methylation, and expression status of decoy receptors in cervical cancer cell lines and apoptotic response to TRAIL- chemo/radiation treatment. +, expressed in normal level; downward arrow indicates decreased expression; -, indicates complete lack of expression; IR, ionizing radiation; Gy, gray.

The cell lines with exogenously over expressed decoy receptors, separately or in combination, should become resistant if their inactivation is critical for TRAIL combination treatment response compared to parental cell lines. Results of these in vitro functional assays, if confirmed, serve as indicators for stratifying cervical cancer or TCL patients for effective TRAIL-based treatment. Because selective induction of apoptosis via TRAIL-R1 pathway has been reported by using an agonistic humanized MAb mapatumumab, also tested is DR4/DR5 monoclonal antibody apoptotic response compared to rhTRAIL.

A large number of molecules are reported to play roles in transmitting signals from DR4/DR5 to apoptotic caspase machinery [25-27]. Because TRAIL-resistance is attributable to attenuated expression of DR4/DR5, the effect of knockdown/restoration of expression of DcR1/DcR2 genes on the levels of DR4/DR5 expression is examined. Sensitization of tumor cells to TRAIL might involve alterations in other downstream effectors such as CFLAR, BCL2 family proteins, and lAPs. Previous studies reported that T-ALL and HTLV-1 positive adult T-cell leukemia cell lines exhibit TRAIL resistance and NF-kB activation may play a role in this resistance ([8, 24]. Anti-apoptotic protein c- FLIP/CFLAR can also be recruited to DISC thus competing with caspase-8 and caspase- 10. Induction of p53, caspase activation, c- FLIPL is examined by RT-PCR and western blot analysis in TRAIL-induced apoptosis in relation to DcR expression status.

EXAMPLE 9:

As shown above, there was a significant correlation between DcR inactivation and response to TRAIL-combination treatment in cultured cervical cancer or T-ALL cell lines. This data can be validated using xenograft model as a preclinical screen for the development of TRAIL therapeutics [28].

Methods are according to Example 1 unless otherwise described.

The tumoricidal effects of TRAIL, selected drugs and their combinations are examined on the two most sensitive TCL cell lines (e.g. Jurkat and MOLT-16) that exhibited methylation and down-regulated expression of one or both DcRs. To test TRAIL- drug resistance, two additional cell lines that exhibit highest resistance in vitro are selected. Tumor cells are subcutaneously seeded in immunocompromised nu/nu mice for their potential to develop tumors after implantation and response to TRAIL combination chemotherapy to suppress tumor growth. One to 3 weeks after implantation, TRAIL alone, selected drug alone, or combination of both, along with controls are administered. Tumor size is monitored by measuring tumor volume and imaging methods. Cell lines stably depleted or reconstituted TNFRSF10D and TNFRSF10C genes alone or together on the same cell lines (see above) are tested to assess the role of inactivation of these genes in tumoricidal activity to TRAIL-combination therapy in vivo. Experiments address (i) contribution of DcR gene inactivation in tumor regression to TRAIL-combination drug therapy, (ii) TRAIL-combination drug chemotherapy induction of cell death sensitivity in cell lines stably depleted DcR expression and similarity to constitutively inactivated cell lines, and (iii) effect of reconstitution of decoy receptor expression on tumor regression in vivo to TRAIL-combination treatment.

It is expected that TNFRSF10C inactivation alone or in combination with TNFRSF10D potentiates the tumoricidal affects with TRAIL-combination therapy in vivo as it does in vitro.

Thus is provided the basis for decoy receptor inactivation as a biomarkerfor effective TRAIL-combination therapy in TCL patients.

EXAMPLE: 10

This examples demonstrates assays to detect 8p deletion or methylation mediated inactivation of DcRs .

Methods are according to Example 1 unless otherwise described.

Detection of 8p deletion by FISH: Fluorescence In Situ Hybridization (FISH) is a simple, robust and most commonly used method for identifying specific chromosome abnormalities. BAC clones spanning TNFRSF10 genes (RP1 1-87501 1 spanning TNFRSF10B, C, D genes; and RP1 1- 109B10 covering TNFRSF10A) are used in a dual color FISH approach using chromosome 8 centromeric probe as control. After appropriate validation (sensitivity and specificity) of the probe, cell lines with known deletion status are tested followed by testing on tissue specimens (frozen, paraffin, tissue microarray, cytological smears). This unequivocally determines the status as 8p deletion or monosomy 8. Samples include invasive cancers as frozen or formalin fixed paraffin embedded individual tumors, TMAs containing invasive tumors, or cytological smears from high-grade CINs and invasive cervical cancer.

Multiplex methylation test for decoy receptors: Commercially available platforms to assess the promoter hypermethylation qualitatively and quantitatively are used. CpG methylation is assayed as a characteristic tumor-related genomic change and the test includes detection of any level of methylation as a correlative marker. A highly specific and robust qualitative assay (e.g., MSP, bisulphite sequencing, and Sequenom quantitative methylation assays) is used different types of tissues with tumor heterogeneity. Multiplex MSP is designed with products under 150 bp in the dense methylated regions, which is determined by bisulphite cloning and sequencing of the cell lines and primary tumors, to amplify both TNFRSF10C and TNFRSF10D promoters. After initial standardization of multiplex MSP on known cell lines and primary tissues for methylation, multiple MSP is applied on 50 tumors each with 8p deletion positive and negative by FISH. Comparison of the single and multiplex MSP establishes the sensitivity and specificity of the multiplex test. This establishes "yes or no" for scoring methylation and its relationship with 8p deletion. Sequenom DNA methylation assay based on matrix- assisted laser desorption/ionization of flight mass spectrometry is also used. This approach provides an accurate test for methylation of DcR genes.

Identification of expression of DcR genes in CC tissues: Because promoter hypermethylation is associated with loss of expression in cancer, the identification of down-regulated transcription or protein provides a means to identify inactivation. This is examined in the entire specimen by RT-PCR/western blot analysis or at individual cell level by in situ methods of detection of mRNA or protein. The IHC is one available method. Several commercially available antibodies are tested against TNFRSF10C and TNFRSF10D on cell lines with known status of methylation and expression on paraformaldehyde fixed cytospin or paraffin-embedded tissue sections. Once the specificity of antibodies is optimized, antibodies are examined on tissues that used in FISH and MSP. Use of DcR1 and DcR2 antibodies in IHC can be according to conventional methods. A standard scoring system of 0-3+ is used. Sensitivity and specificity are established to quantify the levels of expression and correlation with MSP status.

EXAMPLE 11

Cytotoxicity analysis by MTT assay in B-cell lymphoma cell lines [N=10] was measured after exposure to TRAIL-doxorubicin combination treatment (TRAIL, 0.5 pg/ml; Doxorubicin, 15 ng/ml) in relation to TNFRSF10C gene expression. Cell lines included ten B-cell NHL cell lines (Daudi, Raji, SUDHL-4, SU-DHL-4, SU-DHL-5, SU-DHL-8, SU- DHL-10, LY-1, LY-3, WSU, and Farage).

Methods are according to Example 1 unless otherwise described.

Results for B-cell lymphoma cell lines are shown in FIG. 6 . B-cell lymphoma cell lines with decreased expression of TNFRSF10C showed synergistic affect by > 10-fold higher sensitivity to TRAIL -doxorubicin treatment compared to TRAIL alone.

EXAMPLE 12

Cytotoxicity analysis by MTT assay in breast cancer cell lines (N=3) after exposure to TRAIL-combination drugs (TRAIL, 0.5 pg/ml; Doxorubicin, 250 ng/ml; Tamoxifen, 15 mM/ml) to assess cell survival in relation to TNFRSF10C methylation. Cell lines included three breast cancer cell lines (MCF7, Mx-1, and MDA-MB-231).

Methods are according to Example 1 unless otherwise described.

Results for breast cancer cell lines are shown in FIG. 7 . Breast cancer cell lines harboring TNFRSF10C methylation (Mx-1 and MDA-MB-231) were highly sensitive to TRAIL combined with either Tamoxifen or Doxorubicin compared to unmethylated breast cancer cell line (MCF7).

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Claim 1. A method for determining responsiveness to TRAIL-induced apoptosis comprising:

detecting an 8p chromosomal deletion in a sample;

detecting methylation of a promoter of at least one tumor necrosis factor related super family (TNFRSF) decoy receptor; or

detecting expression level of at least one TNFRSF decoy receptor; and

correlating increased TRAIL-induced apoptosis to (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

Claim 2 . The method of claim 1 comprising:

(a) detecting an 8p chromosomal deletion in a sample; and

(b) (i) detecting methylation of a promoter of at least one tumor necrosis factor related super family (TNFRSF) decoy receptor; or

(ii) detecting expression level of at least one TNFRSF decoy receptor.

Claim 3. The method of any one of claims 1-2, wherein the at least one decoy receptor comprises DcR1/TNFRSF10C or DcR2/TNFRSF10D.

Claim 4 . The method of any one of claims 1-3, comprising:

detecting methylation of a promoter of at least two decoy receptors; or

detecting expression level of at least two decoy receptors.

Claim 5. The method of claim 4, wherein the first decoy receptor comprises DcR1/TNFRSF10C and the second decoy receptor comprises DcR2/TNFRSF10D. Claim 6 . The method of any one of claims 1-5, wherein the 8p deletion comprises a deletion at 8p12-p21 .3.

Claim 7. The method of any one of claims 1-6, wherein the 8p deletion comprises an 8.4 Mb minimal region of deletion (MRD) between 22,941-31,338 kb physical interval at 8p12-p21 .3.

Claim 8. The method of any one of claims 1-7, wherein the sample comprises a tumor sample.

Claim 9. The method of any one of claims 1-8, wherein the sample comprises a cervical cancer tumor sample, a T-cell leukemia/lymphoma tumor sample, a B-cell NHL tumor sample, or a breast cancer tumor sample.

Claim 10. The method of any one of claims 1-9, wherein the sample comprises a TRAIL-resistant tumor sample.

Claim 11. The method of any one of claims 1-10, wherein the sample is of a subject.

Claim 12. The method of claim 11, wherein the subject is a mammal.

Claim 13. The method of claim 12, wherein the subject is a human.

Claim 14. The method of any one of claims 1-13, wherein the subject is diagnosed with, or suspected of having, cervical cancer, T-cell leukemia/lymphoma, B-cell NHL, or breast cancer.

Claim 15. The method of any one of claims 1-14, further comprising selecting or modifying a treatment on the basis of detecting (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control. Claim 16. The method of any one of claims 1-15, further comprising administering to the subject a therapeutically effective amount of a TRAIL agonist and an antineoplastic agent upon detecting (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

Claim 17. The method of any one of claims 1-16, further comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a TRAIL agonist, an antineoplastic agent, and a pharmaceutically acceptable carrier or excipient upon detecting (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.

Claim 18. The method of any one of claims 16-17, wherein the TRAIL agonist is selected from the group consisting of Apo2L/TRAIL, HGS-ETR1 MAb, HGS-ETR2 MAb, HGS-TR2J MAb, CS-1008 (TRA-8) MAb, AMG 655 MAb, Apomab MAb, and LBY135 MAb.

Claim 19. The method of any one of claims 16-18, wherein the antineoplastic agent is selected from the group consisting of an alkylating agent, an antimetabolite, a plant alkaloid, a cytotoxic antibiotic, a platinum compound, a methylhydrazine, a monoclonal antibody, a photodynamic/radiation therapy sensitizer, and a protein kinase inhibitor, or a combination thereof.

Claim 20. The method of any one of claims 16-19, wherein the antineoplastic agent is selected from the group consisting of Cyclophosphamide, Chlorambucil, Melphalan, Chlormethine, Ifosfamide, Trofosfamide, Prednimustine, Bendamustine, Busulfan, Treosulfan, Mannosulfan, Thiotepa, Triaziquone, Carboquone, Carmustine, Lomustine, Semustine, Streptozocin, Fotemustine, Nimustine, Ranimustin, Methotrexate, Raltitrexed, Pemetrexed, Pralatrexate, Mercaptopurine, Tioguanine, Cladribine, Fludarabine, Clofarabine, Nelarabine, Cytarabine, Fluorouracil, Tegafur, Carmofur, Gemcitabine, Capecitabine, Azacitidine, Decitabine, Vinblastine, Vincristine, Vindesine, Vinorelbine, Vinflunine, Etoposide, Teniposide, Demecolcine, Paclitaxel, Docetaxel, Paclitaxel poliglumex, Trabectedin, Dactinomycin, Doxorubicin, Daunorubicin, Epirubicin, Aclarubicin, Zorubicin, Idarubicin, Mitoxantrone, Pirarubicin, Valrubicin, Amrubicin, Pixantrone, Bleomycin, Plicamycin, Mitomycin, Ixabepilone, Cisplatin, Carboplatin, Oxaliplatin, Satraplatin, Polyplatillen, Procarbazine, Edrecolomab, Rituximab, Trastuzumab, Alemtuzumab, Gemtuzumab, Cetuximab, Bevacizumab, Panitumumab, Catumaxomab, Ofatumumab, Porfimer sodium, Methyl aminolevulinate, Aminolevulinic acid, Temoporfin, Efaproxiral, Imatinib, Gefitinib, Eriotinib, Sunitinib, Sorafenib, Dasatinib, Lapatinib, Nilotinib, Temsirolimus, Everolimus, Pazopanib, Vandetanib, Afatinib, Masitinib, Toceranib, Amsacrine, Asparaginase, Altretamine, Hydroxycarbamide, Lonidamine, Pentostatin, Miltefosine, Masoprocol, Estramustine, Tretinoin, Mitoguazone, Topotecan, Tiazofurine, Irinotecan, Alitretinoin, Mitotane, Pegaspargase, Bexarotene, Arsenic trioxide, Denileukin diftitox, Bortezomib, Celecoxib, Anagrelide, Oblimersen, Sitimagene ceradenovec, Vorinostat, Romidepsin, Omacetaxine mepesuccinate, Eribulin, Camptothecin, and Tamoxifen.

INTERNATIONAL SEARCH REPORT International application No. PCT/US 12/41267

A . CLASSIFICATION O F SUBJECT MATTER IPC(8) - C 12Q 1/68 (2012.01 ) USPC - 435/6.1 4 According to International Patent Classification (IPC) o r to both national classification and IPC

B . FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) USPC: 435/6.14

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched USPC: 424/573; 435/4; 435/6.1; 435/6.1 1; 435/7.23; 435/334 (see search terms below)

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) WEST (PGPB,USPT,EPAB,JPAB); Google Web; Google Scholar; esp@cenet: TRAIL, apoptosis, methylation, TNFRSF, decoy, chromosome, 8p, DcR1/TNFRSF10C, DcR2/TNFRSF10D, TNFRSF10C/DcR1, TNFRSF10D/DcR2, tumor necrosis, trail apoptosis 8p chromosome deletion, Columbia, Govind Bhagat, Dongxu Xie, Murty Vundavalli

C . DOCUMENTS CONSIDERED TO BE RELEVANT

Category* Citation o f document, with indication, where appropriate, o f the relevant passages Relevant to claim No.

RUBIO-MOSCARDO et al. Characterization of 8p21 .3 chromosomal deletions in B-cell 1-3 lymphoma: TRAIL-R1 and TRAIL-R2 a s candidate dosage-dependent tumor suppressor genes. Blood, 2005, Vol 106, pp 3214-3222; especially abstract; pg 3215, col 2, para 3; pg 3218, col 1, para 2; pg 3219, col 1, para 1

HORNSTEIN e t al. Protein Phosphatase and TRAIL Receptor Genes a s New Candidate Tumor 1-3 Genes o n Chromosome 8p in Prostate Cancer. Cancer Genomics and Proteomics, 2008, Vol 5 , pp 123-136; especially abstract; pg 129, Table III; pg 130, Fig 3

□ Further documents are listed in the continuation o f Box C . □ * Special categories of cited documents: ' ' later document published after the international filing date or priority "A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand to be of particular relevance the principle or theory underlying the invention "E" earlier application or patent but published on or after the international "X document of particular relevance; the claimed invention cannot be filing date considered novel or cannot be considered to involve an inventive "L" document which may throw doubts on priority claim(s) or which is step when the document is taken alone cited to establish the publication date of another citation or other 'Ύ ' document of particular relevance; the claimed invention cannot be special reason (as specified) considered to involve an inventive step when the document is "O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such combination means being obvious to a person skilled in the art "P" document published prior to the international filing date but later than "&" document member of the same patent family

Date o f the actual completion o f the international search Date o f mailing o f the international search report

8 August 2012 (08.08.2012) 2 8 AUG 2012

Name and mailing address o f the ISA/US Authorized officer: Mail Stop PCT, Attn: ISA/US, Commissioner for Patents Lee W. Young P.O. Box 1450, Alexandria, Virginia 22313-1450 PCT Helpdesk: 571-272-4300 Facsimile No. 571-273-3201 PCT OSP: 571-272-7774 Form PCT/1SA^10 (second sheet) (July 2009) INTERNATIONALSEARCH REPORT International application No. PCT/US 12/41267

Box No. II Observations where certain claims were found unsearchable (Continuation of item 2 of first sheet)

This international search report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

Claims Nos.: because they relate to subject matter not required to be searched by this Authority, namely:

□ Claims Nos.: because they relate to parts of the international application that do not comply with the prescribed requirements to such an extent that no meaningful international search can be carried out, specifically:

3. Claims Nos.: 4 2 0 because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box No. Ill Observations where unity of invention is lacking (Continuation of item 3 of first sheet)

This International Searching Authority found multiple inventions in this international application, as follows:

□ As all required additional search fees were timely paid by the applicant, this international search report covers all searchable claims.

As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite payment of additional fees.

As only some of the required additional search fees were timely paid by the applicant, this international search report covers only those claims for which fees were paid, specifically claims Nos.:

4. No required additional search fees were timely paid by the applicant. Consequently, this international search report is restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest I I The additional search fees were accompanied by the applicant's protest and, where applicable, the payment of a protest fee. I I The additional search fees were accompanied by the applicant's protest but the applicable protest fee was not paid within the time limit specified in the invitation. I I No protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet (2)) (July 2009)