WO2013016673A2 - Utilisation de l'expression de dpep1 et tpx2 pour l'évaluation du traitement ou de la durée de survie de patients atteints d'un adénocarcinome du canal pancréatique - Google Patents

Utilisation de l'expression de dpep1 et tpx2 pour l'évaluation du traitement ou de la durée de survie de patients atteints d'un adénocarcinome du canal pancréatique Download PDF

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WO2013016673A2
WO2013016673A2 PCT/US2012/048655 US2012048655W WO2013016673A2 WO 2013016673 A2 WO2013016673 A2 WO 2013016673A2 US 2012048655 W US2012048655 W US 2012048655W WO 2013016673 A2 WO2013016673 A2 WO 2013016673A2
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tpx2
dpepl
tissue
levels
pdac
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WO2013016673A3 (fr
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Syed Perwez HUSSAIN
Geng Zhang
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The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the technology relates to a method for evaluating treatment and/or survival time of patients with pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • PDAC is a devastating malignancy worldwide with a median survival of about six months. Because of the current lack of early detection strategies, more than 80% of patients present with advanced disease at diagnosis, and the overall 5-year survival for PDAC patients is 3-5% (Hezel et al., 2006, Genes Dev 20:1218-49). Gemcitabine is the only first-line chemotherapeutic drug approved for advanced pancreatic. However, single agent Gemcitabine is only moderately effective showing a tumor response rate of about 12% (Oettle et al., 2007, JAMA 297:267-77). The progress with the new treatments for pancreatic cancer has been disappointingly slow.
  • pancreatic cancers The failure of many novel targeted agents used in pancreatic cancer clinical trials may be a result of the molecular heterogeneity found in pancreatic cancers, including somatic mutations and epigenetic changes of oncogenes and tumor suppressor that regulate cell proliferation, survival, and other homeostatic functions (Mahalingam et al., 2009, Expert Opin Emerg Drugs 14:311-28). Therefore, better biomarkers and novel therapeutic targets are indispensable to improve the survival rate of patients with PDAC.
  • pancreatic cancer has defined and validated prognostic markers that are of biological significance in pancreatic cancer (Campagna et al, 2008 Int J Clin Exp Pathol 1 :32-43; Kim et al, 2007, Pancreas 34:325-34; Stratford et al, PLoS Med 7:el000307). Predicting prognosis for patients with pancreatic cancers may identify a subset that could benefit from aggressive intervention including surgery and/or chemotherapy (Garcea 2005) . In addition, the development of a prognostic gene signature might provide insight into molecular subtypes of pancreatic cancer (Yeh 2009).
  • Applicants have discovered that expression levels of dipeptidase 1, renal, [Homo sapies] (DPEPl) (NCBI Gene ID: 1800) and targeting protein for Xklp2, microtubule-associated, homolog (Xenopus laevis) [Homo sapies] (TPX2) (NCBI Gene ID: 22974) are useful as prognostic predictors for PDAC. Moreover, these expression levels are capable of revealing potential targets for new therapies in PDAC cases.
  • One aspect of the description is a method of using expression levels of dipeptidase 1 (DPEPl) for prognosis of pancreatic ductal adenocarcinoma (PDAC) in a patient, comprising: (a) causing a measurement of an expression level of DPEPl in a PDAC tissue of a patient; (b) causing a comparison of the expression level of DPEPl in the PDAC tissue to a reference level of DPEPl ; and (c) causing a prognosis to be made based on the difference between the PDAC levels compared to the normal levels.
  • DPEPl dipeptidase 1
  • PDAC pancreatic ductal adenocarcinoma
  • Another embodiment is the method further comprising: (a) causing a measurement of an expression level of targeting protein for Xklp2 (TPX2) in the PDAC tissue; (b) causing a comparison of the expression level of TPX2 in the PDAC tissue to a reference normal level of TPX2; and causing a prognosis to be made based on the difference between the PDAC levels of both DPEP l and TPX2 measured in the patient to the reference levels of DPEPl and TPX2, respectively.
  • TPX2 Xklp2
  • Another embodiment is the method 2 further comprising the steps of: (a) collecting at least one first sample from a PDAC patient; (b) administering to the subject a cancer treatment; (c) collecting at least one second sample following said treatment; (d) measuring levels of DPEP l or TPX2 in each of the samples; (e) comparing levels of DPEP 1 or TPX2 before and after treatment; and (e) providing a prognosis, wherein the prognosis is based on the difference in the levels of DPEP 1 or TPX2 before and after treatment.
  • These methods are useful to identify high-risk PDAC patients, or to predict an outcome of a therapy for treating PDAC, or to develop a therapy for treating PDAC.
  • prognosis One possible outcome of the prognosis is when the levels of DPEPl and TPX2 are found to change following the treatment, including wherein the prognosis is favorable. Another outcome is wherein the levels of DPEP 1 and TPX2 do not change following the treatment, including wherein the prognosis is unchanged following treatment.
  • RNA may be is extracted from tissue obtained from each sample.
  • RNA may be extracted from tumor tissue, and levels of DPEP l or TPX2 may be measured by quantitative PCR, or by a microarray comprising a multiplicity of single stranded oligonucleotides to measure tissue levels of DPEP l or TPX2.
  • the measurement may comprise contacting said RNA with at least one nucleic acid probe to measure levels of a control RNA.
  • Control RNA may be used, e.g., GAPDH, beta-actin, and 18S RNA.
  • Another aspect of the method uses as sample consisting of a multiplicity of tissue samples, obtained from a multiplicity of subjects and/or a multiplicity of tissue samples.
  • a median or average level of DPEP 1 or TPX2 may be determined from the multiplicity of tissue samples.
  • the method may further involve collecting tissue samples from a multiplicity of normal subjects, determining the median or average level of DPEPl or TPX2 in normal subjects, and providing a prognosis based on a comparison of the levels of DPEPl or TPX2 of the PDAC patient with the median or average level of DPEPl or TPX2 of normal subjects.
  • a reference level of DPEPl or TPX2 may be measured, which may be the median or average level of DPEPl or TPX2 of normal subjects, or in tissues of PDAC patients, including non-cancerous tissue of PDAC patients.
  • the method may involve making a comparison of the level of DPEPl or TPX2 in the first sample of the subject with cancer to the reference level of DPEPl or TPX2, and a further step of providing a prognosis based on the comparison.
  • the prognosis may favorable for an anti-cancer treatment, including where the level of DPEP l or TPX2 in the subject with cancer is statistically the same as the reference level of DPEP l or TPX2.
  • the prognosis may not be favorable for a kinase inhibitor treatment, including where the level of DPEPl or TPX2 in the subject with cancer is statistically substantially different than the reference level of DPEPl or TPX2.
  • Samples may be collected from the subject with cancer at various times before and after anti-cancer treatment.
  • Another aspect of the method involves comparing the level of DPEP l or TPX2 in the first sample with the level of DPEPl or TPX2 in each of the multiplicity of second samples. Again, the prognosis is favorable, including wherein the levels of DPEPl or TPX2 changes over time following treatment, in which an alternate treatment modality is provided.
  • the alternate treatment modality may include administering a kinase inhibitor treatment, and/or a cytotoxic drug.
  • nucleic acid probe used in the analysis is a single stranded nucleic acid, including wherein the single stranded probe hybridizes with the nucleic acid having the sequence of DPEPl (SEQ ID NO: l) or TPX2 (SEQ ID NO:2), or a known genetic variant of said sequence.
  • the method may convert the RNA to cDNA using a reverse transcriptase, including when the cDNA is amplified in a polymerase chain reaction.
  • a computing device may be used, comprising a means to store data to be configured in a prognosis in the form of a report, wherein the prognosis is calculated by comparing a level of expression of DPEPl or TPX2 in tissue of a PDAC patient to a reference value.
  • the computing device may be used to generate as the result of data indicative of DPEP l or TPX2 levels in various samples, the data having been subject to a method of prognosis by the computing device.
  • the report may be displayed.
  • the computing device may also include components for data storage, manipulation, processing, configuration, prognosis, display, and calculation.
  • the computing device may consist of a personal computers, server computers, hand held or laptop devices, smart phones, multiprocessor systems, microprocessor- based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, or distributed computing environments.
  • the computing device may comprise one or more processors and one or more memories, including read only memory (ROM) or random access memory (RAM).
  • the computing device may include disks and drives for writing and reading data, one or more user inputs, and/or a network environment with one or more logical connections to one or more computers, wherein data and information may be sent and received by the computing device and may be manipulated.
  • the computing may be configured to receive data related to one or more measurements of DPEP 1 or TPX2, or store data related to one or more measurements of DPEP 1 or TPX2 in a memory associated with the computing device.
  • the computing device may process the data related to a DPEP 1 or TPX2 based on one or more clinical trials, or is indicative of one or more prognosis, and/or be useful to produce a prognosis of PDAC based on a measurement of a level of expression of DPEP 1 or TPX2.
  • Fig. 1 DPEP1 and TPX2 are associated with cancer-specific mortality in two independent cohorts.
  • Fig. 1A Germany test cohort.
  • Fig. IB Maryland validation cohort.
  • the association of DPEP 1 or TPX2 with patient survival was confirmed in the Maryland validation cohort.
  • PRRl 1 was not associated with survival in the validation cohort.
  • Fig. 1C Combined analysis of Germany test and Maryland validation cohorts.
  • Fig. 2 DPEP1 expressed at lower level and TPX2 at higher level and in pancreatic tumors as compared to adjacent non-tumor tissue.
  • Fig. 2A Germany test cohort. Expression of DPEP 1 and TPX2 in Germany test cohort.
  • Fig. 2B Maryland validation cohort. Validation of DPEP 1 and TPX2 expression in independent Maryland cohort. Dot plots represent gene expression level with relative threshold cycle value (Ct) normalized with endogenous control gene GAPDH. Bars indicate median value. Wilcoxon matched-pairs t-tests P value and tumor: non-tumor ratios (T:N) are indicated in the graphs.
  • Fig. 3. DPEP1 (Fig. 3 A) and TPX2 (Fig. 3B) expression in KPC mutant mice.
  • Total RNA were extracted from frozen pancreatic tissues by using Trizol. Quantitative RT-PCR reactions were performed using Taqman Gene Expression Assays. Mouse GAPDH was used as endogenous control to normalize across the samples.
  • Fig. 4 KRAS and EGF regulated DPEP1 and TPX2 expression through MAPK pathway.
  • Fig. 4A siRNA transfection of Pane- 1 cells. 24 hours after trans fection, gene expression was measured by Taqman RT-PCR. GAPDH was used as endogenous control to normalize across the samples. Log 2 ratio represents the effect of target siRNA compared to negative control siRNA on Panc-1 cells.
  • Fig. 4B EGF and MAPK inhibitor U0126 treatment on Panc-1 cells. Cells were starving in 0.1% FBS for 16 hours before treatments. Cells were treated with RPMI medium containing EGF (20ng/mL) or U0126 (10 ⁇ ) alone for 24 hours.
  • EGF and U0126 Cells were pretreated with U0126 for 1 h before addition of EGF. Control cells remained in RPMI with DMSO. Log2 ratio represents the effect of treatment compared to untreated control cell. Each assay was performed in triplicate. * T-test ⁇ 0.01.
  • Fig. 5 Effect of EGF, AZD6244 and LY294002 on DPEP1 expression.
  • Cells were starved in 0.1% FBS for 16 hours before treatments.
  • Cells were treated with RPMI medium containing EGF (30ng/mL), AZD6244 (1.5 ⁇ ) or LY294002 (1.5 ⁇ ) alone for 24 hours.
  • EGF+AZD6244 or EGF+LY294002 Cells were pretreated with AZD6244 or LY294002 for lh prior to the addition of EGF. Untreated control cells were maintained in RPMI with DMSO.
  • Fig. 5 A Real-time PCR was done to determine DPEP1 mRNA levels.
  • Relative expression of DPEP1 represents the effect of treatment on gene expression compared to untreated control. Data are means ⁇ S.D. from 3 independent experiments. * T-test P ⁇ 0.01.
  • Fig. 5B Western blot showed similar changes at protein level of DPEP 1 after 24 hour treatment.
  • Fig. 5C Western blot demonstrated the efficiency and specificity of AZD6244 and LY294002.
  • DPEP 1 overexpression enhances sensitivity to gemcitabine.
  • DPEP1 overexpressing cells and control cells were analyzed for cellular sensitivity to gemcitabine using Panel (A) and MIApaca2 (B). Overexpession of DPEP1 increased the sensitivity of Panel and MIApaca2 cells to gemcitabine.
  • Control cells are Panel or MIApaca2 cells transfected with GFP control vectors. Cells were treated with Gemcitabine for 96 hours at different doses. The MTS assay was used to quantitate cytotoxicity (cell death) according to the manufacturer's instructions.
  • Relative cytotoxicity (%) was calculated using the formula: [1-(OD 570 of drug treated cells / OD57 0 of untreated cells)] 3 ⁇ 4 100%. Data are means ⁇ S.D. from 3 independent experiments. * T-test P ⁇ 0.01.
  • Fig. 7. DPEP1 overexpression inhibits cell invasion in Panel and MIApaca2 cells.
  • Fig. 7A Cell invasion was analyzed in Panel (upper panel) and MIApaca2 (lower panel) cells using Biocoat matrigel invasion assay. The invaded GFP-positive cells were counted under a fluorescence microscope.
  • Fig. 7B Relative cell invasion is expressed as the ratio of the percent invasion of a test cell over the percent invasion of a control cell. * P ⁇ 0.01.
  • Fig. 9 Combined analysis of Germany test and Maryland validation cohorts, stratified by resection margin status.
  • Fig. 10 cDNA sequence for DPEP1 ( CBI Reference Sequence NM_0044133) (SEQ ID O: l)
  • Fig. 1 1 cDNA sequence for TPX2 (NCBI Reference Sequence NM_012112.4) (SEQ ID NO:2)
  • messenger RNA or "mRNA” means a coding RNA sequence of 5 to 4000 nucleotides in length that can be detected in a biological specimen. Some mRNAs are derived from precursors transcripts processed by nuclear to a mature species. Synthetic DNA complementary to a mRNA (cDNA) can bind to corresponding mRNA or amplified double stranded forms of mRNA and provide a means for detection.
  • cDNA synthetic DNA complementary to a mRNA
  • Biological sample or "body tissue” can be used interchangeably and refer to a fluid isolated from a mammal. Such samples include, but are not limited to, serum, plasma, urine, ascitic fluid, tissue isolated from the PDAC itself. The sample refers to all biological materials isolated from any given subject. In the context of the description such samples include, but are not limited to, the PDAC tissue itself.
  • mRNA Variants are common, for example, among different animal species.. These variants demonstrate a scope of acceptable variation in the sequence of the mRNAs that does not impair function or the ability to detect the mRNA(s). Some modifications may affect the ability to detect the mRNA by qRT-PCR directed against a canonical species, but not by microarray.
  • nucleotide can be used interchangeably and refer to nucleotide sequences of any length, including DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a nucleotide sequence, for example by DNA or RNA polymerase, or by chemical reaction.
  • Nucleic acids may be single stranded or double stranded, or may contain portions of both double and single stranded sequence. A single strand can provide a probe that hybridizes to a target sequence.
  • An "isolated" polynucleotide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in its natural source.
  • An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the specific nucleic acid molecule as it exists in natural cells.
  • “Complement” or “complementary” as used herein in reference to a nucleic acid sequence means Watson and Crick or Hogsteen base pairing between nucleotides or nucleotide analogs.
  • Percent (%) nucleic acid sequence identity means the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in a nucleic acid sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Thymine (T) and uracil (U) may be considered equivalent when comparing DNA and RNA.
  • differential expression means qualitative or quantitative differences in the expression pattern of one or more polynucleotides, including mRNA, in a biological sample. Expression of the one or more polynucleotides may be upregulated, resulting in an increased amount of transcripts, or downregulated, resulting in a decreased amount of transcripts. Expression of the one or more polynucleotides may be upregulated or downregulated in a particular state, such as a disease state, relative to a reference state, such as a normal state, thus permitting comparison of two or more states.
  • the one or more polynucleotides may exhibit a pattern of expression in said body fluid, cell, or tissue that is detectable by standard techniques, including but not limited to expression arrays, quantitative reverse transcriptase PCR, northern analysis, and real-time PCR. Some of the polynucleotides may be expressed in one state but not another.
  • gene includes any polynucleotide sequence or portion thereof with a functional role in encoding or transcribing a protein or regulating other gene expression.
  • the gene may consist of all the nucleic acids responsible for encoding a functional protein or only a portion of the nucleic acids responsible for encoding or expressing a protein.
  • the polynucleotide sequence may contain a genetic abnormality within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences or unique adjacent regions to the gene.
  • tumor refers to malignant neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues.
  • Treatment is an intervention performed with the intention of preventing the development or altering the pathology of a disease or disorder. Accordingly, “treatment” herein refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disease or disorder as well as those in which the disease or disorder is to be prevented.
  • a therapeutic agent may directly decrease the pathology of tumor cells, or render the tumor cells more susceptible to treatment by other therapeutic agents, e.g., radiation and/or chemotherapy.
  • Mature mR As are described herein as useful for having specific nucleotide sequences. However hundreds of genomic variants are known for both DPEP 1 and TPX2 Some of these lead to variant mRNA sequences. While SEQ ID NOS: l and 2 are disclosed herein, variants of these mRNA sequence are hereby included in the current description by reference to the NCBI SNP Database and/or Ensembl (EMBL).
  • EMBL NCBI SNP Database and/or Ensembl
  • mRNA is isolated from a tumor or cancer tissue, the isolated mRNA is converted to cDNA, and amplified.
  • mRNA can be detected by various methods, including reverse transcription polymerase chain reaction (RT-PCR), northern blotting, ribonuclease protection assay (RPA), and in situ hybridization (ISH), or kits such as QuantiGene® (Panomics, Fremont, CA).
  • Kits for isolating RNA, and in particular mRNA, from a biological sample are known and commercially available, such TRIzol® (InvitrogenTM).
  • cDNA can be generated by reverse transcription of isolated mRNA using reverse transcription conventional techniques.
  • mRNA reverse transcription kits are known and commercially available. Examples of suitable kits include, but are not limited to, the TaqMan® and/or High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Specific primers are known and commercially available, for example, from Applied Biosystems (Foster City, Ca), Ambion (Austin, TX), and Qiagen (Valencia, CA).
  • the reverse transcript of the mRNA can be amplified using conventional PCR techniques including, but not limited to, real time PCR. Kits for quantitative real time PCR of RNA and mRNA are known and commercially available.
  • the RNA can be ligated to a single stranded oligonucleotide containing universal primer sequences, a polyadenylated sequence, or adaptor sequence prior to reverse transcriptase and amplified using a primer complementary to the universal primer sequence, poly(T) primer, or primer comprising a sequence that is complementary to the adaptor sequence.
  • a biological sample can be obtained from a single individual or pooled, for example, from a group of individuals suffering from a particular disease or disorder.
  • mRNA can be isolated from the sample by any number of methods, for example, as described herein, and the abundance of one or more mRNA can be determined by any of a number of methods, for example by calculating average Ct values.
  • Amplification curves are checked to verify that Ct values are assessed in the linear range of each amplification plot. Typically, the linear range spans several orders of magnitude.
  • a chemically synthesized version of the mRNA can be obtained and analyzed in a dilution series to determine the limit of sensitivity of the assay, the linear range of quantitation, and to estimate the absolute abundance of the candidate mRNAs measured.
  • Protein expression products of the DPEP1 and TPX2 genes can also be measured and utilized as a means of monitoring expression levels of one or both of these genes. Protein expression levels can be measured using antibodies (e.g rempli immunohistochemistry of tissues) (Millipore, Sigma, R&D Systems, OriGene Antibodies, GenScript, Novus Biologicals).
  • protein mass spectroscopy can be utilized to measure expression levels of one or both of the marker proteins.
  • specific peptides are known for both genes. Biochemical assays for these are known (See EMD Millipore, Sigma-Aldrich, R&D Systems, OriGene, GenScript, Cell Signaling Technology, Enzo Life Sciences, and/or Uscn)
  • Prognosis refers to the probable outcome of a disease, preferably when a patient is diagnosed as having a tumor, and, more preferably, when the patient is diagnosed as having a cancer.
  • the prognosis of a cancer includes the probable outcomes of using a treatment modality, preferably when the treatment involves use of a therapeutic drug.
  • the prognosis can include an outcome in which the cancer is refractory to a possible treatment modality, preferably where the treatment modality will improve the prospects for recovery, increase the chances of survival, reduce the recovery period for the cancer, or minimize the probability of recurrence of the cancer.
  • Most preferably the method of prognosis will identify a suitable treatment modality to improve the probability of a favorable outcome for the patient.
  • a favorable outcome is one in which the cancer patient has at least a 70% chance, and preferably an 80% chance that the cancer will not recur or metastasize within 2, 3, 4, 5, 6, 7, 8, 9, 10 or more months from beginning a therapeutic regime.
  • the treatment regime is related to the level of DPEP1 and/or TPX2 in the blood, serum or bodily fluid of the patient.
  • treatment does not cause a change in levels of DPEP1 and/or TPX2.
  • the prognosis is not favorable, because the cancer may be refractory to treatment.
  • a cancer patient presents with low levels of DPEPl and/or high TPX2 and is refractory to therapy. In this instance, a more aggressive therapy should be considered.
  • Such therapy includes a kinase inhibitor drug and/or treat with a cytotoxic agent.
  • a cytotoxic agent may be chemotherapy or a high dose of radiotherapy.
  • a cancer patient presents with a high level of DPEP l and/or low TPX2. This patient likely will have a favorable prognosis. If their DPEPl level increases and/or TPX2 level decrease when a kinase inhibitor drug is administered, they more likely have better response to conventional chemotherapy, such as Gemcitabine treatment.
  • Whether a level of DPEPl and/or TPX2 is high or low for a specific cancer may be determined by the ordinary skilled worker from a clinical trial conducted with that cancer. In one instance, whether a tissue level of DPEP l and/or TPX2 is low or high depends on the standard value which is predetermined for the specific cancer. In another instance, tissue levels of DPEP l and/or TPX2 are low or high relative to a median or average value obtained from normal, healthy subjects. In another instance, whether tissue levels of DPEPl and/or TPX2 are low depends on an median or average value obtained from different patients with the same cancer. In another instance, the reference value is a median obtained by factoring the value from same cancer patients. The level of DPEPl and/or TPX2 may be normalized by comparing it to the level of a control gene. The skilled person can realize that the reference value may be chosen depending on factors as the normalization method and the control values used.
  • the amount of DPEPl and/or TPX2 in a biological sample can be compared to a reference control, for example a matched sample of normal body tissue, a previously analyzed sample, or a suitable standard control developed for the particular assay.
  • a reference control for example a matched sample of normal body tissue, a previously analyzed sample, or a suitable standard control developed for the particular assay.
  • Kits adapted for the determination of DPEP l and/or TPX2 mRNA expression and prognosis of disease are provided herein.
  • Such kits may include materials and reagents adapted to specifically determine the presence and/or amount of a DPEPl and/or TPX2 mRNA in a sample.
  • the kit can include nucleic acid molecules or probes in a form suitable for the detection of DPEPl and/or TPX2 mRNA.
  • the nucleic acid molecules can be in any composition suitable for the use of the nucleic acid molecules according to the instructions.
  • the kit can include a detection component, such as a microarray, a labeling system, a cocktail of components (e.g., suspensions required for any type of PCR, especially real-time quantitative RT-PCR), membranes, color-coded beads, columns and the like.
  • a detection component such as a microarray, a labeling system, a cocktail of components (e.g., suspensions required for any type of PCR, especially real-time quantitative RT-PCR), membranes, color-coded beads, columns and the like.
  • the kit can include a container, pack, kit or dispenser together with instructions for use.
  • a kit may contain, for example, forward and reverse primers designed to amplify and detect the DPEP1 and/or TPX2 mRNA in biological.
  • Many different PCR primers can be designed and adapted as necessary to amplify one or more mRNA that are differentially expressed in a body fluid and correlate to a particular disease or disorder.
  • the primers are designed to amplify a DPEP 1 and/or TPX2 mRNA.
  • the kit may also contain single stranded oligonucleotide containing universal primer sequences, polyadenylated sequences, or adaptor sequences prior and a primer complementary to said sequences.
  • the mRNA isolated from the biological sample is ligated to the single stranded oligonucleotide containing universal primer sequence, polyadenylated sequence, or adaptor sequence prior to reverse transcription and amplified with said complementary primers.
  • the kit comprises primers that amplify the DPEP 1 and/or TPX2 mRNA.
  • poly- A-tailing is used to generate a sequence that can then be hybridized to a poly-T primer that is used for reverse transcription. See, for example, Shi et al, 2005, BioTechniques 39:519-25.
  • aspects of the present disclosure may take place on a general computing device. Aspects of storing, manipulating, calculating, configuring, or displaying data, prognosing and/or any other computing operations may be performed by a computing device. Examples of well-known computing systems, environments, and/or configurations that may be suitable include, but are not limited to, personal computers, server computers, hand held or laptop devices, smart phones, multiprocessor systems, microprocessor- based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments and the like.
  • An example computing device may comprise one or more processors and one or more memories.
  • the memories may be coupled to the one or more processors.
  • the memories may be coupled to the one or more processors by a system bus, which may be of a type of bus structure known in the art, including a memory bus or memory controller, a peripheral bus, and a local bus using any of a variety of bus architectures.
  • the memories may be read only memory (ROM), random access memory (RAM) and the like.
  • a basic input/output system (BIOS) may be used to transfer data and information between elements within the computing device.
  • a computing device may include disks and drives for writing and reading data.
  • disks and drives may be computer readable media and may have stored thereon instructions that when executed by a processor cause the processor to perform one or more actions included herein.
  • a computing device may be associated with one or more user inputs, such as, for example, a mouse, a keyboard, a voice recorder, touchscreen, joystick, a camera, a medical device with digital output and the like. These and other peripheral input devices may be connected via serial or parallel port to the system bus or any other interface.
  • a monitor, tv, touchscreen, or other type of display device may also be connected to the system bus via an interface such as a video adapter. The same may be true for a printer, a video output, or speakers.
  • a computing device may be in a network environment with one or more logical connections to one or more computers. These may include servers, routers, PCs, network nodes and the like. Data and information may be sent and received by the computing device and may be manipulated.
  • a computing device may be configured to receive data related to one or more measurements of DPEP l and/or TPX2 mRNA.
  • the data related to one or more measurements of DPEP l and/or TPX2 mRNA may be stored in a memory associated with the computing device.
  • the one or more measurements may be stored in a database.
  • a series of trials related to DPEPl and/or TPX2 mRNA mRNA may provide information indicative of the one or more levels of DPEP l and/or TPX2 mRNA based on one or more trials, as well as information indicative of one or more prognoses. Other information indicative of one or more elements related to each prognosis may also be included.
  • the information indicative of the one or more levels of DPEPl and/or TPX2 mRNA mRNA, the information indicative of the one or more prognoses and the information indicative of one or more elements related to the prognosis may be stored in a database.
  • information indicative of one or more elements related to clinical markers of the cancer is provided.
  • the database may be configured as a tool for prognosis.
  • the database may be used as a comparison for data indicative of the levels of DPEP l and/or TPX2 mRNA mRNA.
  • a patient may have their levels of DPEPl and/or TPX2 mRNA mRNA measured. This measurement may be compared to one or more outputs of the database, such as, for example, calculations, similar prognosis factors, patient similarities and the like.
  • the database may be configured to provide a graphical display of the levels of DPEP l and/or TPX2 mRNA and prognosis.
  • the data in the database may be displayed on a display device, or it may be used in one or more calculations.
  • a calculation may comprise a prognosis of a patient associated with the data.
  • the computing device may be configured to provide an output related to the prognosis of a patient as a response to receiving one or more measurements of DPEPl and/or TPX2 mRNA mRNA.
  • computer readable medium may have stored thereon instructions that when executed by a processor may cause the processor to receive data indicative of one or more levels of DPEPl and/or TPX2 mRNA mRNA, manipulate the data, store the data, place the data in a database, subject the data to one or more calculations such as those described herein, and provide a prognosis.
  • a computing device or computer readable medium may be configured to send data indicative of one or more levels of DPEPl and/or TPX2 mRNA; receive, as a response to the sending, an indication of the prognosis of a subject; and display, on a display, the prognosis of the subject.
  • the database above may be used to instruct a researcher or a person in control of choosing an appropriate therapy on the basis of the prognosis of cancer patient.
  • Tumor histopathology was classified according to the World Health Organization (WHO) Classification of Tumor system (Aaltonen et al.,WHO, Int'l Agency for Research on Cancer. Lyon Oxford: IARC Press, 2000). Use of these clinical specimens was approved by the Office of the Human Subject Research (OHSR, Exempt # 4678) at the National Institutes of Health, Bethesda, MD.
  • WHO World Health Organization
  • RNA from frozen tissue samples was extracted using standard TRIzol® protocol. RNA quality was confirmed with the Agilent 2100 Bioanalyzer (Agilent Technologies) before the microarray gene expression profiling. Tumors and paired nontumor tissues from Germany cohort were profiled separately using the Affymetrix GeneChip Human Exon 1.0 ST arrays according to the manufacturer's protocol at LMT microarray core facility at National Cancer Institute, Frederick, MD. All arrays were RMA normalized and gene expression summaries were created for each gene by averaging all probe sets for each gene using Partek ® Genomics Suite 6.5. All data analysis was performed on gene summarized data. The data discussed in this publication have been deposited in National Center for Biotechnology
  • NCBI's Gene Expression Omnibus.
  • High-throughput quantitative RT-PCR of gene expression was performed using 96.96 dynamic array chips from Fluidigm Corporation according to the manufacturer's protocol. Pre-amplification reactions were done in a GeneAmp PCR System 9700 from Applied Biosystems.
  • the IFC Controller HX (Fluidigm Corporation) utilizes pressure to control the valves in the chips and load samples and gene expression assay reagents into the reaction chambers.
  • the BioMark system (Fluidigm Corporation) is a real-time PCR instrument designed to thermal cycle these microfluidic chips and image the data in real time. qRT-PCR reactions in 384 well plates were performed using Taqman Gene Expression Assays on an ASI Prism 7900HT Sequence
  • GAPDH GAPDH were used as the endogenous controls. All assays were performed in quadruplicate or triplicates. For quality control, any samples with a gene cycle value greater than 36 were considered of poor quality and removed. If a tumor or non-tumor sample failed quality control from qRT-PCR that case was removed from the analysis. All the primers for qRT-PCR in the present study were purchased from Applied Biosystems (Table 3). Example 4: Statistical Analysis
  • T-test, Wilcoxon matched-pairs t-tests, and Expression graphs were used to analyze differences in gene expression between tumors and paired non-tumor tissue using Graphpad Prism 5.0 (Graphpad Software Inc, San Diego, California). Correlation analysis and Kaplan-Meier analysis was performed with Graphpad Prism 5.0. Cox Proportional-hazards regression analysis was performed using Stata 1 1 (StataCorp LP, College Station, Texas). Univariate Cox regression was performed on genes and clinical covariates to examine influence of each on patient survival. Final multivariate models were based on stepwise addition and removal of clinical covariates found to be associated with survival in univariate models (P ⁇ 0.05).
  • resection margin status was dichotomized as positive (Rl/2) vs. negative (RO); TNM staging was dichotomized based on non-metastatic ( ⁇ - ⁇ ) vs. metastatic (IIB-IV) disease.
  • Example 5 Cell lines and culture conditions
  • Human pancreatic carcinoma cell lines PANC-1 (ATCC CRL-1469), were obtained from American Type Culture Collection ATCC (Rockville, MO, USA). Cells were maintained in GIBCO® RPMI Media 1640 supplemented with GlutaMAXTM-l (Invitrogen), penicillin-streptomycin (50 IU/ml and 50 mg/ml, respectively), and 10% (v/v) fetal calf serum (FCS). Cells were incubated at 37°C in a humidified atmosphere with 10% CO 2 . Human recombinant EGF was purchased from BO Biosciences. MEK-1/2 inhibitor U0126 was purchased from Cell Signaling.
  • LY294002 was purchased from Cell Signaling and dissolved in DMSO (dimethyl sulfoxide) to make 50mM stock solution.
  • AZD6244 were purchased from ChemieTek and dissolved in DMSO to make 40 mM stock solutions. All siRNAs and transfection reagent were purchased from Dharmacon.
  • Si-DPEPl ON-TARGETplus siRNA for OPEP 1 (J-005852-05-0010); Si-Kras: SMART pool siRNA for Kras (J-005069-00-0005); and Si-TPX2: SMART pool siRNA for TPX2 (L-010571-00-00005).
  • Example 6 Transgenic mouse model
  • KPC mice spontaneously develop PDAC and have a dramatically shortened median survival of approximately 5 months as compared with their control littermates (wild-type Pdx-l-Cre mice).
  • the majority oiKPC animals develop cachexia and abdominal distension, highly reminiscent of clinical findings seen in the human disease.
  • Example 8 DPEP 1 and TPX2 are associated with cancer-specific mortality
  • RNA quantification method Therefore, expression levels of these 53 genes were measured by qRT-PCR in tumor and non-tumor tissues in the Germany test cohort.
  • the top differentially expressed genes between tumor and non-tumor were TPX2, DCBLD2 and ANLN which were significantly increased in tumors (T:N ratio>2.0, O.01), whereas CDOl, DPEPl, C7, ALDH1A1 and NR3C2 which were significantly decrease in tumors (T:N ratio ⁇ 0.2, O.01).
  • DPEPl expression was decreased (T: N ratio of 0.1 in test cohort and 0.16 in validation cohort, O.01) and TPX2 expression was increased (T: N ratio of 2.14 in test cohort and 2.2 in validation cohort, O.001) in tumor tissue, compared to non-tumor tissues in both cohorts (Fig. 2).
  • TPX2 may be a useful biomarker to identify a subset of patients with poor prognosis in the resection margin negative patients, for which micro-metastasis is not yet detectable by current clinical methods.
  • Multivariate Cox proportional hazards analysis was used to further evaluate the association of DPEP1 or TPX2 expression in tumors with prognosis in the combined cohort (Table 2). The dichotomized DPEP1 or TPX2 expression values were not associated with resection margin status or tumor stage.
  • results presented herein provide evidence that DPEP1 and TPX2 are prognostic biomarkers, independent of resection margin status and other clinical covariates, in multiple cohorts of PDAC. Furthermore, an association of these two biomarkers with RAS/MAPK signaling pathway provides insights in developing efficient multi-target treatments for PDAC.
  • Example 9 DPEPl and TPX2 expression regulated by activated KRAS mutant.
  • Pancreatic adenocarcinoma is driven by activating mutation in the KRAS oncogene, which is present in more than 95% of all cases (Almoguera et al., 1988, Cell 53 :549- 54; Morris et al, Nat Rev Cancer 10:683-95).
  • KRAS possibly could play a role in the observed alterations of DPEPl and TPX2 level in PDAC. Therefore, DPEP 1 and TPX2 expression were compared between KPC transgenic mice and the littermate wild-type control mice.
  • KRAS expression in Panc-1 cells was knocked down by siRNA. Since these cells endogenously express mutant KRAS 012® , KRAS knockdown may affect expression of DPEP l and TPX2.
  • DPEPl was found to be increased and TPX2 was decreased when KRAS expression was knocked down 90% (Fig. 4A).
  • direct knocking-down of TPX2 also increased DPEPl level, indicating that TPX2 itself could be an upstream regulator for DPEPl .
  • Example 10 MEK-MAPK pathway is required in the regulation of TPX2 and DPEP l expression.
  • EGF epidermal growth factor
  • MAPK pathway is an important mediator in the regulation of TPX2 and DPEP 1 expression by mutant KRAS or growth factor such as EGF in pancreatic cancer.
  • DPEPl is a membrane bound dipeptidase and may be involved in the degradation of surrounding extracellular matrix components (Mciver et al, 2004,), a mechanism that would facilitate the invasion processes of tumors. DPEPl is also implicated in the metabolism of glutathione, an important antioxidant (24, 26). Reduce DPEP l expression leads to less glutathione with increased oxidative stress, which may promote carcinogenesis (Klaunig et al, 1998, Environ Health Perspect 106:Suppl 1 :289-95).
  • the TPX2 gene is located on the long arm of chromosome 20, at position
  • TPX2 overexpression positively correlated with tumor grade and stage, with lympho-metastasis and was associated with poor survival rate (Ma et al, 2006, Clin Cancer Res 12: 1121-27; Li B et al. Brain Res 1352: 200-07).
  • immunohistochemical staining of a tissue microarray showed that TPX2 protein level was higher in pancreatic tumors compared with their normal counterparts (36).
  • Our study give the direct evidence from quantitative RT-PCR that TPX2 mRNA level was elevated in pancreatic tumor compared with surrounding non-tumor tissues.
  • TPX2 prognostic significance
  • Activating mutations of KRAS are the earliest consistently detected abnormality in the development of pancreatic cancer.
  • the MAPK pathway is one of the most thoroughly analyzed downstream pathways of activated RAS (Lewis et al, 1998, Adv Cancer Res 74:49-
  • RAS-RAF-MEK-MAPK cascade plays a central role in mediating growth factor-triggered signals (Giehl, 2000; Roberts et al, 2007, Oncogene 26:3291-310).
  • EGF has been found to promote pancreatic cancer cell migration and invasion by activating MAPK pathway and most human pancreatic carcinoma cells are characterized by overexpression of EGF and its receptor (EGFR) (Giehl, 2000; Friess et al, 1996, J Mol Med
  • MAPK signaling pathway was found to be required in the regulation of DPEPl and TPX2 expression.
  • a significant increase ( ⁇ 2 fold) in DPEPl gene expression was also found when cells were treated with AZD6244 alone (PO.01) in 24h (Fig. 5A).
  • LY294002 alone had no effect on DPEPl expression.
  • pancreatic cancer treatment which can increase DPEP 1 and reduce TPX2 level, may improve patients' survival.
  • the results described herein suggest a number of possible targets for pancreatic cancer treatment, such as MAPK pathway and TPX2 (Fig. 4C).
  • TPX2 is a microtubule-associated protein that plays a central role in mitotic spindle formation and therefore cell cycle progression (Gruss et al, 2001, Cell 104:83- 93).
  • TPX2 is another putative mediator of cell cycle alteration in response to the inhibition of MAPK pathway.
  • Example 12 DPEPl sensitizes pancreatic cancer cells to Gemcitabine
  • Multivariate analysis is adjusted for cohort membership, TPX2, DPEPl, and resection margin status. Multivariate analysis used stepwise addition and removal of clinical covariates found to be associated with survival in Univariate model and final models include only those covariates that were significantly associated with survival (P ⁇ 0.05).

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Abstract

L'invention concerne un procédé d'utilisation des niveaux d'expression de la dipeptidase 1 (DPEP1) pour l'évaluation du traitement et/ou de la durée de survie de patients atteints d'un adénocarcinome du canal pancréatique (PDAC), comprenant le dosage des niveaux d'expression de DPEP1 et de la protéine de ciblage pour Xklp2 (TPX2) dans le tissu de PDAC du patient, et l'évaluation du traitement et/ou de la survie, sur la base des niveaux de PDAC de DPEP1 et TPX2 mesurés chez le patient.
PCT/US2012/048655 2011-07-27 2012-07-27 Utilisation de l'expression de dpep1 et tpx2 pour l'évaluation du traitement ou de la durée de survie de patients atteints d'un adénocarcinome du canal pancréatique WO2013016673A2 (fr)

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