WO2016057629A1 - Procédés et thérapeuthiques concernant la protéine r-spondine humaine et la protéine de récepteurs couplés aux protéines g contenant des motifs répétés riches en leucine - Google Patents

Procédés et thérapeuthiques concernant la protéine r-spondine humaine et la protéine de récepteurs couplés aux protéines g contenant des motifs répétés riches en leucine Download PDF

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WO2016057629A1
WO2016057629A1 PCT/US2015/054419 US2015054419W WO2016057629A1 WO 2016057629 A1 WO2016057629 A1 WO 2016057629A1 US 2015054419 W US2015054419 W US 2015054419W WO 2016057629 A1 WO2016057629 A1 WO 2016057629A1
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sample
protein
subject
cancer tumor
tumor
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Gregory A. MICHELOTTI
Brittany Bohinc HENDERSON
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Duke University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • sequence listing is filed with the application in electronic format only and is incorporated by reference herein.
  • sequence listing text tile "DU4410PCT_ST25.txt.” was created on October 5, 2015, and is 56,803 bytes in size.
  • This invention relates to a method for stratifying risk associated with metastasis of certain cancers having elevated expression of human R-spondin (RSPO) protein or leucine-rich repeat-containing G protein-coupled receptor (LGR) proteins.
  • RSPO R-spondin
  • LGR leucine-rich repeat-containing G protein-coupled receptor
  • the Wnt/ -catenin signaling pathway plays an important role in stem cell proliferation, tissue differentiation, and cellular polarity. Dysregulation of Wnt/p-catenin signaling contributes to the development of human papillary thyroid cancer (PTC), potentiating cancer stem cell proliferation, loss of cellular polarity and thyroid microarchitecture, tumor dedifferentiation, extracapsular spread, and angioinvasion.
  • PTC human papillary thyroid cancer
  • CSCs cancer stem cells
  • LGR4 and LGRS are orphan receptors of the G-protein-coupled receptor superfamily that are specifically expressed on CSCs. Recently, LGR4 and LGR5 have been shown to bind with high affinity to a family of secreted peptide ligands known as R-spondins (RSPOl-4).
  • R-spondin (RSPO) protein family is a small family of four secreted growth factors. The four paralogs share 40-60% pairwise amino acid sequence identity and are predicted to share substantial structural homologies.
  • RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands.
  • a characteristic feature of all four RSPO members is their ability to activate ⁇ -catenin signaling and enhance WNT -mediated ⁇ -catenin activation.
  • lymph node recurrence is routinely seen in patients with papillary thyroid cancer after their initial surgery. In fact, a majority of these patients are followed with lifetime serial neck ultrasonography to evaluate for lymph node recurrence and many undergo repeat surgery, which is not only costly, but has increase incidence of surgical complications.
  • the typical diagnostic and treatment regimen for human papillary thyroid cancer is as follows: 1) perform thyroid ultrasound to identify lymph nodules; 2) based on size, appearance, a history of external beam radiation, a family history of cancer, and combinations thereof, select suspicious nodules for further examination; 3) acquiring a biopsy sample from one or more of the suspicious nodules, typically by fine needle aspiration biopsy; 4) cytopatho logically categorize the biopsy sample as benign, malignant/suspicious, or
  • Surgical extent is based on perioperative thyroid ultrasound lymph node mapping and is also based on surgeon preference. Note that central lymph nodes, those where the cancer is likeliest to metastasize first, cannot be seen preoperatively because they are covered by the thyroid itself, so preoperative ultrasound only samples lymph nodes in the lateral neck.
  • this disclosure provides a pre-surgical method of stratifying risk of metastasis of a thyroid cancer tumor in a subject suspected of having the thyroid cancer tumor.
  • the method includes measuring a human R-spondin (RSPO) protein concentration in a serum sample from the subject or a leucine -rich repeat-containing G protein-coupled receptor (LG ) and/or RSPO protein concentration in a fine needle aspiration (FNA) sample or a thyroid tissue sample from the subject, and stratifying the risk of metastasis of the thyroid cancer tumor based on the measuring.
  • RSPO human R-spondin
  • LG leucine -rich repeat-containing G protein-coupled receptor
  • FNA fine needle aspiration
  • this disclosure provides a method of monitoring the efficacy of a therapeutic treatment for a subject having cancer.
  • the method includes the following: measuring, in a sample acquired at a first time to provide a reference measurement, a first human R-spondin (RSPO) protein concentration in a first serum sample or a first fine needle aspiration (FNA) sample acquired from the subject at the first time or a first solid tumor sample acquired from a solid cancer tumor of the subject at the first time or a first leucine-rich repeat-containing G protein-coupled receptor (LGR) protein concentration in the FNA sample or the first solid tumor tissue sample acquired from the solid cancer tumor of the subject at the first time; measuring, in a sample acquired at a second time to provide an efficacy measurement, a second human RSPO protein concentration in a second serum sample or a second FNA sample acquired from the subject at the second time or a second solid tumor sample acquired from the solid cancer tumor of the subject at the second time or a second LGR protein concentration in a second FNA
  • the therapeutic treatment is administered at least partially between the first time and the second time.
  • this disclosure provides a method of prognosis of metastatic disease in a subject having a solid cancer tumor.
  • the method includes measuring a human R- spondin (RSPO) protein concentration in a serum sample acquired from the subject or a leucine- rich repeat-containing G protein-coupled receptor (LGR) protein concentration in a solid tumor tissue sample acquired from the solid cancer tumor of the subject, and assessing a risk of metastatic disease based on the measuring.
  • RSPO human R- spondin
  • LGR leucine- rich repeat-containing G protein-coupled receptor
  • this disclosure provides a method of treating a thyroid cancer in a subject.
  • the method includes administering to the subject a therapeutically effective amount of an active agent, the active agent substantially silencing expression of a leucine-rich repeat- containing G protein-coupled receptor (LGR) protein or ⁇ -catenin.
  • LGR G protein-coupled receptor
  • this disclosure provides a method of treating a solid cancer tumor in a subject.
  • the method includes determining if the solid cancer tumor possesses a mutation to the BRAF gene, and if the solid cancer tumor possesses the mutation to the BRAF gene, administering to the subject a therapeutically effective amount of an active agent, the active agent substantially silencing expression of a leucine -rich repeat-containing G protein- coupled receptor (LGR) protein or ⁇ -catenin.
  • LGR leucine -rich repeat-containing G protein- coupled receptor
  • this disclosure provides a pre-surgical method of stratifying risk of a thyroid cancer tumor in a subject suspected of having thyroid cancer.
  • the method includes the following: optionally performing a traditional thyroid cancer treatment cycle on the subject; in parallel with or in place of the traditional thyroid cancer treatment cycle, measuring a human R- spondin (RSPO) protein concentration in a serum sample from the subject or a leucine-rich repeat-containing G protein-coupled receptor (LGR) and/or a human RSPO protein concentration in a fine needle aspiration (FNA) sample or a thyroid tissue sample from the subject;
  • RSPO human R- spondin
  • LGR leucine-rich repeat-containing G protein-coupled receptor
  • FNA fine needle aspiration
  • the serum sample, the FNA sample, or the thyroid tissue sample categorizing the serum sample, the FNA sample, or the thyroid tissue sample as benign or malignant/suspicious; in cases where the biopsy sample, the serum sample, the FNA sample, the thyroid tissue sample, or a combination thereof is categorized as malignant/suspicious, recommending surgical intervention.
  • the traditional thyroid cancer treatment cycle includes the following: performing an ultrasound on the patient to identify suspicious nodules based on a characteristic of the nodules selected from the group consisting of a size, an appearance, a history of external beam radiation, a family history of thyroid cancer, and combinations thereof; acquiring a biopsy sample from at least one of the suspicious nodules; cytopathologically categorizing the biopsy sample as benign, malignant/suspicious, or indeterminate; and in cases categorized as indeterminate, re-categorizing the indeterminate biopsy sample, using a genetic panel conducted on the biopsy samples, as benign or malignant/suspicious.
  • Figure 1A is a plot of mRNA expression levels for TPC- 1 cells treated with varying concentrations of RSPO 1 , as described in Example 1.
  • Figure 1C shows a Western blot confirming increased LGR5 after treatment with RSP03.
  • Figure 2 includes data indicating niclosamide inhibition of Wnt signaling in KTC-1 cells.
  • KTC-1 cells were plated at 30% confluency and treated with vehicle (DMSO), 0.5 uM, or 2.0 uM niclosamide after Fugene transfection with TOPFLASH or FOPFLASH plasmids.
  • vehicle DMSO
  • 0.5 uM 0.5 uM
  • 2.0 uM niclosamide after Fugene transfection with TOPFLASH or FOPFLASH plasmids.
  • TOPFLASH plasmid has three Tcf response elements upstream of the thymidine kinase promoter (Tk) and luciferase gene that bind to active ⁇ -catenin to drive transcription of Wnt target genes.
  • FOPFLASH is a negative control plasmid that contains mutated Tcf response elements in the same vector backbone. Renilla luciferase plasmid was co-transfected to control for transfection efficiency. At the indicated time, cells were harvested, lysed in hypotonic buffer, and
  • FIG. 3 provides evidence that shRNA-mediated decrease in LGR5 expression results in inhibition of cell migration, as described in Example 2.
  • TPC1 cells harboring stable shR A sequence for either control scrambled shRNA (top) or targeting human LGR5 (bottom) were subjected to a wound healing assay for 24 and 48 h. Representative images are shown. Briefly, cells were grown to 90% confuence at which time cells were removed with a P200 tip. Images were then acquired at Oh, 24h, and 48h and analyzed by Olympus software to measure ability of the cells to repair the wound.
  • Figure 4 is a plot of quantified cellular migration with results expressed as mean ⁇
  • Figure 5 includes data representing an increased expression of LGR4 and LGR5 in the KTC-1 cell line compared with the TPC-1 cell line, as described in Example 3.
  • Figure 6 includes data representing an increased expression of LGR5 in human frozen
  • Figure 7 includes data representing an increased expression of RSPOl, RSP02, and RSP03 in cancer cell lines as compared to normal thyroid cells, as described in Example 4.
  • Figure 8 includes data indicating that LGR5 expression is higher in PTC tumor tissue that was BRAFV600E positive as compared to PTC tumoral tissue that was mutation negative, as described in Example 5.
  • recitation of a value between 1 and 10 or between 2 and 9 also contemplates a value between 1 and 9 or between 2 and 10. Ranges identified as being "between" two values are inclusive of the end- point values. For example, recitation of a value between 1 and 10 includes the values 1 and 10.
  • This disclosure relates generally to methods and therapeutics for improving the diagnosis and treatment of certain types of cancer. Specifically, this disclosure relates to thyroid cancer, such as papillary thyroid cancer, metastatic disease, and cancers having the BRAFV600E mutation. Pre-Surgical Methods of Stratifying Risk of Metastasis of a Thyroid Cancer Tumor in a Subject Suspected of Having the Thyroid Cancer Tumor
  • This disclosure provides a pre-surgical method of stratifying a risk of metastasis of a thyroid cancer tumor in a subject suspected of having the thyroid cancer tumor.
  • the method can include measuring a human R-spondin (RSPO) protein concentration in a serum sample from the subject or a leucine-rich repeat-containing G protein-coupled receptor (LGR) and/or a human RSPO protein concentration in a fine needle aspiration (FNA) sample or a thyroid tissue sample from the subject.
  • the method can also include stratifying the risk of metastasis of the thyroid cancer tumor based on the measuring.
  • the method includes measuring both a human RSPO protein concentration and a LGR protein concentration.
  • stratifying the risk of metastasis can be further based on: a comparison of the measuring to a normal control value of human RSPO protein concentration in a normal serum sample or a normal control value of LGR protein concentration or human RSPO protein concentration in a normal FNA sample or a normal thyroid tissue sample of at least 100 patients not having a second thyroid cancer tumor; or a comparison of the measuring to an overexpressed value of human RSPO protein concentration in an overexpressed serum sample or an overexpressed value of LGR protein concentration or human RSPO protein concentration in an overexpressed FNA sample or an overexpressed thyroid tissue sample of at least 100 patients having a third thyroid cancer tumor.
  • the terms second and third are simply to distinguish from a tumor in the patient of interest, and are not intended to imply that there are any specific number of tumors.
  • the human RSPO protein can be selected from the group consisting of RSPOl, RSP02, RSP03, RSP04. In certain aspects, the human RSPO protein is RSP02 or RPS03.
  • RSPOl can have the sequence set forth in SEQ ID NO: 1 (Isoform 1), the sequence set forth in SEQ ID NO: 2 (Isoform 2), or the sequence set forth in SEQ ID NO: 1 with deletions at positions 146-208 (Isoform 3). In some aspects, RSPOl can have a M ⁇ V substitution at position 150 of Isoform 1 or a M ⁇ V substitution at position 123 of Isoform 2.
  • RSP02 can have the sequence set forth in SEQ ID NO: 3 (Isoform 1), the sequence set forth in SEQ ID NO: 3 with deletions at positions 1-67 (Isoform 2), or the sequence set forth in SEQ ID NO: 4 (Isoform 3). In some aspects, RSP02 can have a L ⁇ P substitution as position 186 of Isoforms 1 or 2 or a L ⁇ P substitution as position 122 of Isoform 3.
  • RSP03 can have the sequence set forth in SEQ ID NO: 5 (Isoform 1) or the sequence set forth in SEQ ID NO: 6 (Isoform 2). In some aspects, RSP03 can have a C ⁇ R substitution at position 41, a R ⁇ I substitution at position 170, or a
  • RSP04 can have the sequence set forth in SEQ ID NO: 7 (Isoform 1) or the sequence set forth in SEQ ID NO: 7 with deletions as positions 137-198 (Isoform 2).
  • RSP04 can have a Q ⁇ R substitution at position 65, a C ⁇ F substitution at position 95, a R ⁇ Q substitution at position 106, a C ⁇ R substitution at position 107, a C ⁇ Y substitution at position 118, or a combination thereof.
  • the LGR protein can be selected from the group consisting of LGR4, LGR5, and LGR6. In certain aspects, the LGR protein is LGR5.
  • LGR4 can have the sequence set forth in SEQ ID NO: 8 (Isoform 1) or the sequence set forth in SEQ ID NO: 8 with deletions at positions 62-85 (Isoform 2).
  • LGR4 can have a S ⁇ G substitution at position 22, a P ⁇ H substitution at position 172, a S ⁇ G substitution at position 215, a N ⁇ S substitution at position 233, a L ⁇ R substitution at position 247, a F ⁇ S substitution at position 292, a L ⁇ P substitution at position 433, a A ⁇ V substitution at position 480, a L ⁇ S substitution at position 668, a L ⁇ I substitution at position 681, a R ⁇ G substitution at position 684, a T ⁇ M substitution at position 709, a D ⁇ G substitution at position 844, or a combination thereof.
  • LGR5 can have the sequence set forth in SEQ ID NO: 9 (Isoform 1) or the sequence set forth in SEQ ID NO: 9 with deletions at positions 143-214 (Isoform 3) or deletions at positions 263-286 (Isoform 2).
  • LGR5 can have a R ⁇ H substitution at position 90, a L ⁇ W substitution at position 212, a H ⁇ R substitution at position 383, a V ⁇ A
  • LGR6 can have the sequence set forth in SEQ ID NO: 10 (Isoform 1), SEQ ID NO: 11 (Isoform 2), or SEQ ID NO: 12 (Isoform 3).
  • LGR6 can have a N ⁇ K substitution at position 267 (Isoform 3), position 128 (Isoform 1), or position 215 (Isoform 2), a A ⁇ S substitution at position 516 (Isoform 3), position 377 (Isoform 1), or position 464 (Isoform 2), a CSPTP ⁇ MISPT substitution at positions 545-549 (Isoform 3), positions 406-410 (Isoform 1), or positions 493-497 (Isoform 2), a V ⁇ A substitution at position 592 (Isoform 3), position 453 (Isoform 1), or position 540 (Isoform 2) a G ⁇ C substitution at position 725 (Isoform 3), position 586 (Isoform 1), or position 673 (I
  • the thyroid cancer tumor can be a papillary thyroid cancer tumor.
  • stratifying the risk can produce a negative predictive value (NPV) of at least 90%.
  • NPV negative predictive value
  • the method can further comprise treating the subject on the basis of the stratifying step.
  • the method can include optionally performing a traditional thyroid cancer treatment cycle on the subject.
  • the traditional thyroid cancer treatment cycle can include performing an ultrasound on the patient to identify suspicious nodules based on a characteristic of the nodules selected from the group consisting of a size, an appearance, a history of external beam radiation, a family history of thyroid cancer, and combinations thereof.
  • the traditional thyroid cancer treatment cycle can also include acquiring a biopsy sample from at least one of the suspicious nodules.
  • the traditional thyroid cancer treatment cycle can further include cytopathologically categorizing the biopsy sample as benign, malignant/suspicious, or indeterminate.
  • the traditional thyroid cancer treatment cycle can include, in cases categorized as indeterminate, re -categorizing the indeterminate biopsy sample, using a genetic panel conducted on the biopsy samples, as benign or malignant/suspicious.
  • the method can include, in parallel with or in place of the traditional thyroid cancer treatment cycle, measuring a human R-spondin (RSPO) protein concentration in a serum sample from the subject or a leucine-rich repeat-containing G protein-coupled receptor (LGR) and/or a human RSPO protein concentration in a fine needle aspiration (FNA) sample or a thyroid tissue sample from the subject.
  • the method can further include categorizing the serum sample, the FNA sample, or the thyroid tissue sample as benign or malignant/suspicious.
  • the methods can also include, in cases where the biopsy sample, the serum sample, the FNA sample, the thyroid tissue sample, or a combination thereof is categorized as malignant/suspicious, recommending surgical intervention.
  • RSPO and/or LGR positivity can be used to direct extent of surgery (hemithyroidectomy vs. total thyroidectomy vs. total thyroidectomy +/- node dissection).
  • This disclosure also provides a method of monitoring the efficacy of a therapeutic treatment for a subject having cancer.
  • the method can include measuring, in a sample acquired at a first time to provide a reference measurement, a first human R-spondin (RSPO) protein concentration in a first serum sample or a first fine needle aspiration (FNA) sample acquired from the subject at the first time or a first solid tumor sample acquired from a solid cancer tumor of the subject at the first time or a first leucine-rich repeat-containing G protein-coupled receptor (LGR) protein concentration in the FNA sample or the first solid tumor tissue sample acquired from the solid cancer tumor of the subject at the first time.
  • RSPO human R-spondin
  • FNA fine needle aspiration
  • LGR leucine-rich repeat-containing G protein-coupled receptor
  • the method can further include measuring, in a sample acquired at a second time to provide an efficacy measurement, a second human RSPO protein concentration in a second serum sample or a second FNA sample acquired from the subject at the second time or a second solid tumor sample acquired from the solid cancer tumor of the subject at the second time or a second LGR protein concentration in a second FNA sample acquired from the subject at the second time or a second solid tumor tissue sample acquired from the solid cancer tumor of the subject at the second time.
  • the method can also include comparing the efficacy measurement with the reference measurement, wherein the therapeutic treatment is administered at least partially between the first time and the second time.
  • the method can include measuring both the human RSPO protein
  • the first human RSPO protein or the second human RSPO protein can be selected from the group consisting of RSPO 1, RSP02, RSP03, and RSP04. In certain aspects, the first human RSPO protein or the second human RSPO protein can be RSP02 or RSP03. In certain aspects, the first human RSPO protein and the second human RSPO protein can be the same.
  • the first LGR protein or the second LGR protein can be selected from the group consisting of LGR4, LGR5, and LGR6. In certain aspects, the first LGR protein or the second LGR protein can be LGR5. In certain aspects, the first LGR protein and the second LGR protein can be the same.
  • the cancer can be thyroid cancer.
  • the thyroid cancer can be papillary thyroid cancer.
  • the method can further comprise altering the therapeutic treatment on the basis of the comparing step.
  • This disclosure further provides a method of prognosis of metastatic disease in a subject having a solid cancer tumor.
  • the method can include measuring a human R-spondin (RSPO) protein concentration in a serum sample from the subject or a leucine-rich repeat- containing G protein-coupled receptor (LGR) and/or a human RSPO protein concentration in a fine needle aspiration (FNA) sample or a solid tumor sample acquired from the solid cancer tumor of the subject.
  • the method can also include assessing a risk of metastatic disease based on the measuring.
  • the method can include measuring the human RSPO protein concentration of the LGR protein concentration.
  • assessing the risk of metastatic disease can be further based on: a comparison of the measuring to a normal control value of human RSPO protein concentration in a normal serum sample or a normal control value of LGR protein concentration or human RSPO protein concentration in a normal FNA sample or a normal tissue sample of at least 100 patients not having a second solid cancer tumor; or a comparison of the measuring to an overexpressed value of human RSPO protein concentration in an overexpressed serum sample or an overexpressed value of LGR protein concentration of human RSPO concentration in an overexpressed FNA sample or an overexpressed solid tumor sample of at least 100 patients having a third solid cancer tumor.
  • assessing the risk of metastatic disease can be further based on: a comparison of the measuring to a normal control value of human RSPO protein concentration in a normal serum sample or a normal control value of LGR protein concentration or human RSPO protein concentration in a normal FNA sample or a normal tissue sample of at least 100 patients not having a second thyroid cancer tumor; or a comparison of the measuring to an overexpressed value of human RSPO protein concentration in an overexpressed serum sample or an overexpressed value of LGR protein concentration of human RSPO concentration in an overexpressed FNA sample or an overexpressed solid tumor sample of at least 100 patients having a third thyroid cancer tumor.
  • the human RSPO protein can be selected from the group consisting of RSPOl, RSP02, RSP03, RSP04. In certain aspects, the human RSPO protein can be RSP02 or RPS03.
  • the LGR protein can be selected from the group consisting of LGR4, LGR5, and LGR6. In certain aspects, the LGR protein can be LGR5.
  • the metastatic disease can originate from a solid cancer tumor.
  • the solid cancer tumor can be a thyroid cancer tumor, a breast cancer tumor, a colorectal cancer tumor, a lung cancer tumor, a melanoma tumor, an ovarian cancer tumor, a pancreatic cancer tumor, a prostate cancer tumor, a uterine cancer tumor, a bladder cancer tumor, a kidney cancer tumor, a stomach cancer tumor, a head and neck cancer tumor, or a combination thereof.
  • the solid cancer tumor can be a thyroid cancer tumor, a colorectal cancer tumor, a melanoma tumor, or a combination thereof.
  • the method can further comprise treating the subject on the basis of the assessing step.
  • assessing the risk can produce a negative predictive value (NPV) of at least 90%.
  • This disclosure further provides a method of treating a thyroid cancer in a subject.
  • the method can include administering to the subject a therapeutically effective amount of an active agent.
  • the active agent can substantially silence expression of a leucine-rich repeat- containing G protein-coupled receptor (LGR) protein or ⁇ -catenin.
  • LGR leucine-rich repeat- containing G protein-coupled receptor
  • This disclosure also provides a method of treating a solid cancer tumor in a subject.
  • the method can include determining if the solid cancer tumor possesses a mutation to the BRAF gene.
  • the mutation to the BRAF gene can be BRAFV600E.
  • the method can include
  • the active agent can substantially silence expression of a leucine-rich repeat-containing G protein-coupled receptor (LGR) protein or ⁇ -catenin.
  • LGR leucine-rich repeat-containing G protein-coupled receptor
  • primers are utilized.
  • the sequences for the primers are identified as follows: ⁇ -actin forward (SEQ ID NO: 13) and reverse (SEQ ID NO: 14); LGR5 forward (SEQ ID NO: 15) and reverse (SEQ ID NO: 16); RSPOl forward (SEQ ID NO: 17) and reverse (SEQ ID NO: 18); RSP02 forward (SEQ ID NO: 19) and reverse (SEQ ID NO: 20); RSP03 forward (SEQ ID NO: 21) and reverse (SEQ ID NO: 22); Axin2 forward (SEQ ID NO: 23) and reverse (SEQ ID NO: 24); and ⁇ -catenin forward (SEQ ID NO: 25) and reverse (SEQ ID NO: 26).
  • LGR5 signaling has been linked with Wnt/p-catenin expression using in vitro cellular drug manipulations.
  • PTC cell lines After treating PTC cell lines with RSPOl, an increase in ⁇ -catenin was demonstrated up to 25 -fold by mRNA and in a dose-dependent manner. Briefly, PTC cells were grown to 60% confluence. Cells were then serum starved for 18 h (0.2% FBS), followed by treatment with either RSPO or niclosamide for up to 72 h. Total RNA was harvested in situ by the Trizol method (Life Technologies) and interrogated for mRNA levels by qPCR.
  • DMSO vehicle
  • 0.5 uM 0.5 uM
  • 2.0 uM niclosamide after Fugene transfection with TOPFLASH or FOPFLASH plasmids.
  • TOPFLASH plasmid has three Tcf response elements upstream of the thymidine kinase promoter (Tk) and luciferase gene that bind to active ⁇ -catenin to drive transcription of Wnt target genes.
  • FOPFLASH is a negative control plasmid that contains mutated Tcf response elements in the same vector backbone. Renilla luciferase plasmid was co-transfected to control for transfection efficiency. At the indicated time, cells were harvested, lysed in hypotonic buffer, and luminescence quantified using commercially available kit (Promega).
  • Treatment of human PTC cell line with niclosamide significantly reduced Wnt signaling at 24 and 48 hours at the 0.5 uM dose. (Fig. 2).
  • LGR5 expression was knocked down in thyroid cancer cell lines by stably integrating LGR5 -specific shRNA. Briefly, RNA interference technology was employed to significantly decrease LGR5 expression using commercially validated shRNA sequences (Origene). TPC1 cells were transiently transfected with plasmids harboring short hairpin (sh)RNA sequences specific for human LGR5 in addition to a puromycin resistance gene. Cells were then selected for stably integrated plasmid through puromycin selection (5 ug/ml) over 10 passages. In parallel, control stable cell lines were created harboring non-specific shRNA sequences that are not known to target any human gene. Results revealed a dramatic phenotype upon knockdown of LGR5 expression.
  • sh short hairpin
  • RNA was isolated from the NThy-ori 3-1, TPC-1, and KTC-1 cell lines, and the expression levels of LGR5 (normalized to ⁇ -actin gene expression, expressed as mean fold-over Nthy-ori 3-1 for each gene, with n 3 independent isolations), and the results are shown in Fig. 5.
  • LGR5 positivity was also associated with male sex (more aggressive disease is seen in males) and with tumor vascular invasion (higher risk for metastasis) (Table 1).
  • LGR5 positivity having a sensitivity of 95.5% (95% CI 88.8%-98.7%), a specificity of 60.9% (95% CI 48.4- 72.4%), a PPV of 75.7% (95% CI 66.6%-83.3%), and a NPV of 91.3% (95% CI 79.2%-97.5%) for predicting lymph node metastases.
  • LGR5 tumor over-expression was found to be strongly associated with lymph node metastasis (p ⁇ 0.0001) and features of tumor aggressiveness (including increased tumor size, lymph node number, higher AJCC stage, increased vascular invasion, extrathyroidal extension, capsular invasion and macroscopic tumor invasion). In several cases, there was striking intra-tumoral heterogeneity. In patients with positive lymph nodes, tumor stained positive for LGR5 in the lymph nodes as well as in the original tumor. Tumors that were heterogeneous for LGR5 with associated locoregional metastases to lymph nodes had strong LGR5 positivity within the lymph node metastatic foci themselves, suggesting that LGR5 signaling may play an important role in tumor aggressiveness.
  • Example 5 LGR5 Expression in BRAFV600E Mutation-Positive Tumors.
  • RSP02 increased serum concentrations were associated with the following:
  • RSP02 immunohistochemistry was also performed and correlated with LGR5 tumor status.
  • RSP02 and LGR5 positivity were highly positively correlated (p ⁇ 0.0001, both by likelihood ratio and Fisher's exact testing).
  • the odds ratio for positivity if LGR5 positive was 4.9875 (95% CI: 2.27-10.94).
  • RSP02 by IHC was also associated with the following:
  • the odds ratio of a tumor having associated lymph node metastases was 6.844 (95% CI 3.35-13.97) if the tumor was both LGR5 and RSP02 positive (p ⁇ 0.001);
  • thyroid nodule F As "diagnostic for thyroid cancer” or “suspicious for thyroid cancer” (Bethesda Categories 5/6).
  • relevant clinical data will be collected from the chart (specifically, age, sex, race, BMI, TSH level, free T4/free T3, TPOAbs, TRAbs, co-morbidities) and patients will be given a thyroid cancer risk assessment questionnaire.
  • Pre-operative ultrasound nodule characteristics will be reviewed (specifically, size of the nodule, echotexture, suspicious US features etc).
  • a peri-operative lymph node mapping by ultrasound will be performed assessing bilateral levels II-VI for abnormal lymphadenopathy prior to surgery. All abnormal lymph nodes will be FNA'd (cytology +/- Tg needle washout) according to usual clinical standard of care. Results will direct extent of surgery as per usual clinical standard of care.
  • the patient will be consented for prophylactic ipsilateral central lymph node dissection for assessment of central node metastatic evaluation. If abnormal lymph nodes are found at surgery, extent of neck dissection will be left to the surgeon's clinical discretion. Final pathology report and tumor characteristics will be collected. Upon surgical removal, FNA will be performed directly on the surgical tumor specimen and preserved for protein/DNA/RNA assessment prior to being sent for final pathology.
  • All patients referred to the clinic for FNA biopsy of a thyroid nodule will be asked to consider participation in the research study. If consented, clinical data from the medical chart will be collected including: age, sex, ethnicity, BMI, TSH, free T3, free T4, TPOAbs, TRAbs/TSII, comorbidities, and thyroid cancer risk assessment questionnaire.
  • a pre-operative ultrasound for lymph node mapping in the lateral neck will be performed per clinical standard of care.
  • FNA will be performed directly on the surgical specimen immediately after surgery for collection of protein, RNA, and DNA. Prophylactic ipsilateral central neck dissection for lymph nodes will be performed on all study participants.
  • lymph nodes are found intraoperatively and/or pre-operatively by ultrasound, full extent of lymph node dissection will be left to the clinical expertise of the treating surgeon. All cytology and pathology reports will be collected in the final analysis. Patients that undergo surgical intervention will be approached for tumor donation to the tissue bank and collection of additional slides from the pathology tissue block for laboratory-based LGR5 and RSP02 immunohistochemical staining of the tumor.
  • the primary outcome will be: whether up-regulation of LGR5/RSPO detected in an FNA sample can pre-operatively predict lymph node metastases. If our hypothesis is correct, preoperative assessment for LGR5/RSPO may help direct extent of surgery (i.e. TT only, TT + central neck dissection, and/or TT + central and modified ipsilateral/contralateral neck dissections). Secondary outcomes will be: whether extent of up-regulation of these tumor markers can preoperatively predict BRAFV600E mutational status and/or additional features of tumor aggressiveness as assessed by pathology.
  • Results will be analyzed initially using descriptive statistics. Comparison between groups will be done using chi square tests for proportions, and t-tests of ANOVA procedures for continuous variables. Regression analysis will be performed to identify independent outcome predictors Other inferential statistical analysis will be conducted as appropriate. JMP7 software will be used for analysis.

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Abstract

La présente invention concerne des procédés et des thérapeutiques concernant l'expression de la protéine R-spondine humaine ou de la protéine des récepteurs couplés aux protéines G contenant des motifs répétés riches en leucine dans les cellules cancéreuses. Les procédés comprennent un procédé pré-chirurgical de stratification d'un risque de métastase d'une tumeur de cancer de la thyroïde chez un sujet chez qui l'on soupçonne une tumeur cancéreuse de la thyroïde, un procédé de surveillance de l'efficacité d'un traitement thérapeutique pour un sujet ayant un cancer, un procédé de pronostic de maladie métastasique chez un sujet ayant une tumeur cancéreuse solide, un procédé de traitement d'un cancer de la thyroïde, un procédé de traitement d'une tumeur cancéreuse solide chez un sujet, et un procédé pré-chirurgical de stratification d'un risque de tumeur de cancer de la thyroïde chez un sujet chez qui l'on soupçonne une tumeur cancéreuse de la thyroïde.
PCT/US2015/054419 2014-10-07 2015-10-07 Procédés et thérapeuthiques concernant la protéine r-spondine humaine et la protéine de récepteurs couplés aux protéines g contenant des motifs répétés riches en leucine WO2016057629A1 (fr)

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CN110462404A (zh) * 2017-01-12 2019-11-15 理论科学株式会社 测试受试者患有胰腺癌的可能性的方法
CN115792247A (zh) * 2023-02-09 2023-03-14 杭州市第一人民医院 蛋白组合在制备甲状腺乳头状癌风险辅助分层系统中的应用

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WO2009126380A2 (fr) * 2008-03-05 2009-10-15 Singulex, Inc. Procédés et compositions pour une détection hautement sensible de molécules
WO2010016766A2 (fr) * 2008-08-08 2010-02-11 Koninklijke Nederlandse Akademie Van Wetenschappen Anticorps reconnaissant un lgr5 et/ou un lgr6 humain endogène
US20100131432A1 (en) * 2008-11-17 2010-05-27 Kennedy Giulia C Methods and compositions of molecular profiling for disease diagnostics
US20110275065A1 (en) * 2010-05-07 2011-11-10 Ranju Ralhan Methods and compositions for the diagnosis and treatment of thyroid cancer
US20130336970A1 (en) * 2007-07-02 2013-12-19 Oncomed Pharmaceuticals, Inc. Compositions and Methods for Treating and Diagnosing Cancer
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WO2009126380A2 (fr) * 2008-03-05 2009-10-15 Singulex, Inc. Procédés et compositions pour une détection hautement sensible de molécules
WO2010016766A2 (fr) * 2008-08-08 2010-02-11 Koninklijke Nederlandse Akademie Van Wetenschappen Anticorps reconnaissant un lgr5 et/ou un lgr6 humain endogène
US20100131432A1 (en) * 2008-11-17 2010-05-27 Kennedy Giulia C Methods and compositions of molecular profiling for disease diagnostics
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110462404A (zh) * 2017-01-12 2019-11-15 理论科学株式会社 测试受试者患有胰腺癌的可能性的方法
CN110462404B (zh) * 2017-01-12 2023-06-02 理论科学株式会社 测试受试者患有胰腺癌的可能性的方法
CN115792247A (zh) * 2023-02-09 2023-03-14 杭州市第一人民医院 蛋白组合在制备甲状腺乳头状癌风险辅助分层系统中的应用
CN115792247B (zh) * 2023-02-09 2023-09-15 杭州市第一人民医院 蛋白组合在制备甲状腺乳头状癌风险辅助分层系统中的应用

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