ES2849965A1 - METHOD FOR PREDICTING OR FORECASTING RESPONSE TO CANCER TREATMENT - Google Patents

Abstract

Biomarker to predict or predict the response to cancer treatment, specifically, to the treatment of sarcomas, preferably osteosarcoma.

Description

METHOD FOR PREDICTING OR FORECASTING THE RESPONSE TO

CANCER TREATMENT

FIELD OF THE INVENTION

The present invention is within the field of medicine, and relates to a biomarker and a method for predicting or predicting response to cancer treatment, preferably sarcoma treatment.

STATE OF THE ART

Primary bone sarcomas, which mainly include osteosarcoma, chondrosarcoma, and Ewing's sarcoma, are a group of rare tumors of mesenchymal or ectodermal origin that constitute <0.2% of all malignant neoplasms in adults. However, high-grade osteosarcomas and Ewing's sarcomas mainly affect children and young adults (<20 years) where they account for approximately 3.5% and 2%, respectively, of all solid cancers. Although advances in surgery and multi-agent chemotherapy have markedly improved the prognosis of bone sarcoma, the 5-year survival rate remains at 60-70% for patients with localized disease, and as low as 20-30% for patients with metastases. The poor prognosis of bone sarcomas is partly related to their resistance to conventional chemotherapy and radiation therapies. This makes bone and cartilage cancers the third leading cause of cancer death in children and adolescents in the United States. Therefore, there is an urgent need for new therapies, biomarkers (diagnostic, prognostic or therapeutic) and a greater understanding of the mechanisms behind the chemo and radioresistance of bone sarcomas.

Transforming growth factor (TGF) -p is a pleiotropic cytokine that regulates proliferation, angiogenesis, carcinogenesis, and immune responses. TGF-p is produced as an inactive complex consisting of the mature TGF-p homodimer bound to the latency-associated peptide (LAP). A conformational change of LAP induced by integrins, proteases, reactive oxygen species, or low pH, releases TGF-p which can then bind to its receptors and induce signaling through canonical (SMAD) and non-canonical pathways. Although TGF-p initially acts as a tumor suppressor, in later stages of carcinogenesis it promotes the tumor growth and metastasis through induction of epithelial to mesenchymal transition (EMT), promotion of tumor cell proliferation, inhibition of antitumor immune response, and recruitment / induction of cancer-associated fibroblasts in the tumor microenvironment . Importantly, recent data suggest that TGF-P may also play a role in the chemo and radioresistance of tumors. Predominant glycoprotein A repeats (GARPs), also known as leucine-rich repeats containing 32 (LRRC32), bind latent TGF-P1 to the surface of activated regulatory T cells (Tregs) and B cells where they promote activation. of TGF-P1 and is essential for its suppressive functions and isotype change, respectively. Recent studies have shown that GARP expression in human lung cancer and colorectal tumors is associated with a worse prognosis. However, the expression and role of GARP in mesenchymal neoplasms is unknown. Mesenchymal stem cells (MSCs) are multipotent non-hematopoietic cells that can differentiate into osteoblasts, chondroblasts, and adipocytes. MSCs represent an important component of the bone marrow (BM) where they regulate hematopoiesis and participate in bone homeostasis. The abundant evidence may contain oncogenic events during SCD differentiation that can lead to bone sarcomas. In fact, some sarcomas include surface markers and gene expression profiling with murine MSCs and BM-MSCs can lead to osteosarcoma through spontaneous transformation and genetic modification. However, it is unknown whether GARP is expressed. in bone sarcoma cells and the possible effects of GARP on its tumorgenicity and chemo / radiosensitivity are unknown.

DESCRIPTION OF THE FIGURES

Figure 1. Several sarcoma cell lines express GARP and their silencing blocks the proliferation of these lines. (A) GARP mRNA expression data in various cancer cell lines retrieved from the CCLE database. RMA log2 mRNA expression values are shown as box plots and separated in ascending order from the median. Outliers are displayed as solid circles. (B) Expression of GARP in mRNA in primary BM-MSCs and various cancer cell lines. Data are shown as the mean (SD), relative to GARP expression in BM-MSCs. (C) Summary of GARP expression (% relative to isotype control) in primary BM-MSC and sarcoma cell lines selected bone. Data is shown as the mean (SD) of three individual stains. (D) The silencing of GARP in BM-MSCs and in osteosarcoma cell lines G292, T1-73 and SAOS-2 inhibits its proliferation. The proliferation (cell index) of untransduced cells (NT), LV-CTRL, GARPKO1 and GARPKO2 was measured in real time for 7 days. Data are shown as the mean (SD) of experimental duplicates. A representative experiment of 3. (E) Graphs of three individual experiments is shown showing the mean (SD) of proliferation as cell index at 160 hours divided by the corresponding cell index at 10 hours. * = P <0.05; *** = P <0.001. (F) Silencing GARP in osteosarcoma cell lines induces apoptosis cell death. Apoptosis in G292 NT, LV-CTRL, GARPKO1 and GARPKO2 cells (left graph), T1-73 (center graph) and SA0S-2 (right graph) was measured by staining the cells with 7AAD / Annexin V and analyzed cells by flow cytometry. Data are shown as the mean (SD) of three independent experiments. * = P <0.05.

Figure 2. GARP promotes the proliferation of bone sarcoma cell lines through the activation of TGF-p. (A) GARP overexpression in bone sarcoma cell lines. Representative dot plots of G292 untransduced (NT) and overexpressing GARP (GARP ++) (upper panel), SAOS-2 (central panel) and RD-ES (lower panel). The staining of the corresponding isotype is represented with vertical lines in all graphs. (B) GARP overexpression increases the proliferation of bone sarcoma cell lines. Growth curves of G292, SAOS-2 and RD-ES NT, GARP ++ and EGFP (EGFP ++) overexpressing cells were generated by plotting the number of accumulated cells versus time. Data are shown as the mean (SD) of three individual experiments. * = P <0.05. (C) The increase in the number of EGFP ++ and GARP ++ cells relative to NT cells at the end of each experiment is shown as the mean (SD) of three individual experiments. * = P <0.01. (D) GARP ++ sarcoma cells produce more active TGF-p compared to NT cells. G292, SAOS-2 and RD-ES NT and GARP ++ cells were co-cultured with SBE-HEK293 cells for 24 hours and then the luciferase activity was analyzed. The increase in TGF-p activity of GARPP ++ cells is relativized to NTs for each cell line.

Data are represented as the mean (SD) of three independent experiments. * = P <0.05, ** = P <0.01. (E) Increased proliferation of GARP ++ sarcoma cells is TGF-p dependent. G292, SAOS-2 and RD-ES NT cells were cultured and GARP ++ in the presence or absence of SB431542 or the anti-TGF-pi / 2/3 antibody (11D1) for 3 days and then the cells were counted. The increase in the number of cells is relativized to the NTs for each cell line. Data are shown as the mean (SD) of three independent experiments. * = P <0.05; ** = P <0.01.

Figure 3. GARP increases the growth of tumors from G292 cells in vivo . (A) G292 NT (n = 10), LV-CTRL (n = 6), GARPKO2 (n = 10) and GARP ++ (n = 10) cells were injected subcutaneously into NSG mice and tumor growth was followed for 70 days. Tumor weight and width were measured weekly using an automated caliper and tumor volume was calculated as described in Materials and Methods. Data is shown as the mean (SD). * = P <0.05 GARP ++ vs NT. (B) Representative image of isolated NT and GARP ++ tumors at the end of the experiment (upper panel). The volumes (left graph) and weights (right graph) of the isolated tumors were measured as described in materials and methods. Data are shown as the mean (SD) with open (NT) and orange (GARP ++) circles representing individual tumors. (C) Representative image of isolated LV-CTRL and GARPKO tumors at the end of the experiment (upper panel). The volumes (left graph) and weights (right graph) of isolated tumors were measured as described in materials and methods. Data are shown as the mean (SD) with black (LV-GARP) and blue (GARPKO2) circles representing individual tumors. Tumor cell proliferation was analyzed by Ki67 immunohistochemical staining in NT (n = 10) and GARP ++ (n = 10) tumors. Data are shown as the mean (SD) with open (NT) and orange (GARP ++) circles representing individual tumors. * = P <0.05. (E) Ki67 staining in LV-CTRL (n = 6) and GARPKO2 (n = 3) tumors. Data are shown as the mean (SD) with black (LV-CTRL) and blue (GARPKO2) circles representing individual tumors. (F) Activation of the TGF-p signaling pathway was analyzed by phospho-SMAD3 immunohistochemistry in NT and GARP ++ tumors. Data are shown as the mean (SD) with open (NT) and orange (GARP ++) circles representing individual tumors. * = P <0.05.

Figure 4. GARP overexpression in bone sarcoma cell lines makes them more resistant to death induced by etoposide and doxorubicin. G292, SAOS-2 and RD-ES cells were untransduced (NT), overexpressing GARP (GARP ++) and overexpressing EGFP (EGFP ++) to different concentrations of etoposide in vitro. (A) Cell viability was measured using CelltiterBlue and represented as% control (cells without etoposide). Data are shown as the mean (SD) of three individual experiments. * = P <0.05; ** = P <0.01; *** = P <0.001. (B) G292, SAOS-2 and RD-ES NT and GARP ++ cells were subjected to 80 pM (G292) or 10 pM (SAOS-2 and RD-ES) of etoposide, with or without SB431542 or the anti-TGF- pi / 2/3 (11D1). Cell viability was analyzed using CelltiterBlue and represented as% control (cells without etoposide). Data are shown as the mean (SD) of three individual experiments. * = P <0.05. (C) G292, SAOS-2 and RD-ES NT and GARP ++ cells were subjected to 2.5 pM doxorubicin with or without SB431542. Cell viability was analyzed using CelltiterBlue and represented as% control (cells without doxorubicin). Data are shown as the mean (SD) of three individual experiments. * = P <0.05.

Figure 5. GARP overexpression in bone sarcoma cell lines makes them more resistant to radiation-induced death and DNA damage.

Susceptibility to irradiation was analyzed by a clonogenicity test. SAOS-2 (A) and RD-ES (B) NT and GARP ++ cells were seeded at low density in 6-well plates and cultured with or without SB431542 as described in materials and methods. Cells were irradiated at 2, 4, and 8 Gy. After 2 weeks, the colonies were fixed, stained with crystal violet, and counted as described in materials and methods. Data are represented as the survival fraction (relative to non-irradiated cells) and are shown as the mean (SD) of three independent experiments. * = P <0.05; *** = P <0.001 GARP ++ vs. NT, # = P <0.05; ## = P <0.01; ### = P <0.001 GARP ++ SB431542 vs. GARP ++. (C, D) SAOS-2 (C) and RD-ES (D) NT and GARP ++ cells were grown with or without SB431542, irradiated at 4 Gy, and analyzed for g-H2AX by flow cytometry. Data are shown as the mean (SD) of 3 independent experiments. (E, F) SAOS-2 (E) and RD-ES (F) NT and GARP ++ cells were grown with or without SB431542, irradiated at 4 Gy, and analyzed for g-H2AX using the Imagestream flow cytometer. The number of positives g-H2AX per nucleus is shown as the mean (SD). A representative experiment of 3 is shown.

Figure 6. Cell lines derived from tumors of patients with osteosarcoma and chondrosarcoma express GARP. (A) Tumor cells derived from patients were extracted from two osteosarcoma tumors (OST-3 and OST-4) and from one chondrosarcoma tumor (T-CDS17). These cell lines were stained for GARP expression on the surface and analyzed by flow cytometry. Representative staining is shown. (B) The proliferation of OST-4 NT cells was analyzed, LVCTRL and GARPKO2 using the xCelligence Real Time Proliferation Analyzer System. Data are shown as the mean (SD) of experimental duplicates. A representative experiment of two is depicted.

Figure 7. Immunohistochemical analysis and impact on patient survival of GARP expression in sarcoma tissue samples. (A) Representative examples of the indicated sarcoma types showing high and low GARP staining. Scale bars: 500 gm (red) or 100 gm (black). (B) Distribution of sarcoma cases (N = 89) according to their GARP expression level in the categories of the indicated patient characteristics and the clinicopathological parameters of the tumor (a more exhaustive description is shown in Table S2) . The P values are shown. (C) Kaplan-Meier cumulative survival curves classified by GARP protein expression in the sarcoma patient cohort. P values were estimated using the log-rank test.

Figure 8. GARP expression and response to treatment - Pilot study. (A) Information regarding the type of tumor, treatment received and corresponding response. CR: complete response, SE: stable disease, PR: partial response, PD: progressive disease, GIST: gastrointestinal stromal tumor, S. Ewing: Ewing's sarcoma. (B) Dichotomous analysis of patients with complete response vs. RC RP PD EE. SUPPLEMENTARY FIGURE LEGENDS

Figure S1. GARP silencing in bone sarcoma cell lines. G292, T1-73 and SAOS-2 cell lines were transduced with LV-CTRL, LV-GARPKO1 and LV-GARPKO2 as described in materials and methods. Four days later, GARP expression was measured by flow cytometry. Data are shown as the mean (SD) of two (T1-73) or three (G292, SAOS-2) independent experiments.

Figure S2. GARP overexpression in G-292, SAOS-2, and RD-ES cells makes them more resistant to etoposide-induced apoptosis compared to NT cells. G292, SAOS-2 and RD-ES NT and GARP ++ cells were exposed to different concentrations of etoposide for 24 hours, stained for Annexin V / 7AAD, as described in supplementary materials and methods, and analyzed by flow cytometry. Data are shown as the mean (SD) of at least three individual experiments. * = P <0.05, ** = P <0.01.

Table S1. Distribution of sarcoma cases (N = 89) according to their GARP expression levels in the categories of indicated patient characteristics and clinicopathological parameters. The P values are shown.

Table S2. Univariate and multivariate Cox analysis of GARP expression and clinicopathological parameters, including tumor necrosis, tumor grade, tumor count, and tumor type.

DESCRIPTION OF THE INVENTION

We show, in the examples of the invention, that GARP is expressed in BM-derived MSCs and in several established and recently isolated primary patient-derived bone sarcoma cell lines. Also, the silencing of GARP inhibited its proliferation, while the overexpression of GARP increased its proliferation in vitro and in vivo, through a TGF-p-dependent mechanism. Bone sarcoma cells overexpressing GARP were more resistant to in vitro cell death induced by etoposide, doxorubicin, and irradiation, which again depended on increased TGF-p activation. They also show that GARP is overexpressed in human osteo, chondro, and pleomorphic sarcomas and is associated with a significantly worse prognosis. Importantly, GARP expression in human sarcoma tumors was associated with lower survival and a trend towards GARP expression was observed that has independent prognostic value. Therefore, GARP could serve as a therapeutic target for the treatment of bone sarcoma and also as a possible prognostic and predictive marker of the response of bone sarcomas to chemotherapy and radiotherapy.

Therefore, GARP could serve as a marker with therapeutic, prognostic and predictive value for tumors, and more specifically, bone sarcoma.

Therefore, a first aspect of the invention relates to GARP for use as a biomarker to predict or predict the response to cancer treatment in an individual.

GARP (also called Glycoprotein A Repetitions Predominant, Leucine Rich Repeat Containing 32, Transforming Growth Factor Beta Activator LRRC32, Leucine-Rich Repeat-Containing Protein 32, D11S833E, Garpin, LRRC32) is understood as a key growth factor regulator. transformant beta (TGFB1, TGFB2 and TGFB3) that controls the activation of TGF-beta by keeping it in a latent state during storage in the extracellular space. It is specifically associated through disulfide bonds with the latency-associated peptide (LAP), which is the regulatory chain of TGF-beta, and regulates dependent activation of TGF-beta integrin. Able to compete with LTBP1 to bind to the TGF-beta LAP regulatory chain. It controls the activation of TGF-beta-1 (TGFB1) on the surface of activated regulatory T cells (Tregs). Required for epithelial fusion during palatal development by regulating the activation of TGF-beta-3 (TGFB3) (by similarity).

In the context of the present invention, "GARP" is also defined by a nucleotide or polynucleotide sequence, which constitutes the sequence of the "GARP" gene, and which would comprise various variants from:

a) Nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,

b) Nucleic acid molecules whose hybrid complementary chain with the polynucleotide sequence of a),

c) Nucleic acid molecules whose sequence differs from a) and / or b) due to the degeneracy of the genetic code.

d) Nucleic acid molecules that encode a polypeptide comprising the amino acid sequence with an identity of at least 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2, in the that the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the GARP protein. Other possible nucleotide sequences encoding GARP include, but are not limited to, SEQ ID NO: 1.

Gene ID: 2615

Gene location: 11q13.5

SEQ ID NO: 1 GGTGGGGCAGCTGAGCGGCCTGCTCCTCCTCGGGCCGTGACCCCGGGGTCTGGCGCGGGGTGGGACC CGGGGGCGGGTTTGCGCAAAATGTGCCGAGACTGCCCGGGAGAGGAGCTGCGGCCGCGCTGAGCCA GGTGGACAAGAAGGTCrcGTGCCAGGTTCTGGGCCTGCrcCAGGTCCCC _1- CGGTGCrcCCGCCAGACA CTGAGACCCTTGATCTATCTGGGAACCAGCTGCGGAGTATCCTGGCCTCACCCCTGGGCTTCTACACAG CACTTCGTCACCTGGACCTGAGCACCAATGAGATCAGCTTCCTCCAGCCAGGAGCCTTCCAGGCCCTGA CCCACCTGGAGCACCTCAGCCTGGCTCACAACCGGCTGGCGATGGCCACTGCGCTGAGTGCTGGTGGC CTGGGCCCCCTGCCACGCGTGACCTCCCTGGACCTGTCTGGGAACAGCCTGTACAGCGGCCTGCTGGA GCGGCTGCTGGGGGAGGCACCCAGCCTGCATACCCTCTCACTGGCGGAGAACAGTCTGACTCGCCTCA CCCGCCACACCTTCCGGGACATGCCTGCGCTGGAGCAGCTTGACCTGCATAGCAACGTGCTGATGGAC ATCGAGGATGGCGCCTTCGAGGGTCTGCCCCGCCTGACCCATCTCAACCTCTCCAGGAATTCCCTCACCT GCATCTCCGACTTCAGCCTCCAGCAGCTGCGGGTGCTAGACCTGAGCTGCAACAGCATCGAGGCCTTTC AGACGGCCTCCCAGCCCCAGGCTGAGTTCCAGCTCACCTGGCTTGACCTGCGGGAGAACAAACTGCTC CATTTCCCCGACCTGGCCGCGCTCCCGAGACTCATCTACCTGAACTTGTCCAACAACCTCATCCGGCTCC CCACAGGGCCACCCCAGGACAGCAAGGGCATCCACGCACCTTCCGAGGGCTGGTCAGCCCTGCCCCTC TCAGCCCCCAGCGG GAATGCCAGCGGCCGCCCCCTTTCCCAGCTCTTGAATCTGGATTTGAGCTACAAT GAGATTGAGCTCATCCCCGACAGCTTTCTTGAGCACCTGACCTCCCTGTGCTTCCTGAACCTCAGCAGAA ACTGCTTGCGGACCTTTGAGGCCCGGCGCTTAGGCTCCCTGCCCTGCCTGATGCTCCTTGACTTAAGCC ACAATGCCCTGGAGACACTGGAACTGGGCGCCAGAGCCCTGGGGTCTCTGCGGACGCTGCTCCTACAG GGCAATGCCCTGCGGGACCTGCCCCCATACACCTTTGCCAATCTGGCCAGCCTGCAGCGGCTCAACCTG CAGGGGAACCGAGTCAGCCCCTGTGGGGGGCCAGATGAGCCTGGCCCCTCCGGCTGTGTGGCCTTCTC CGGCATCACCTCCCTCCGCAGCCTGAGCCTGGTGGATAATGAGATAGAGCTGCTCAGGGCAGGGGCCT TCCTCCACACCCCACTGACTGAGCTGGACCTTTCTTCCAATCCTGGGCTGGAGGTGGCCACGGGGGCCT TGGGAGGCCTGGAGGCCTCCTTGGAGGTCCTGGCACTGCAGGGCAACGGGCTGATGGTCCTGCAGGT GGACCTGCCCTGCTTCATCTGCCTCAAGCGGCTCAATCTTGCCGAGAACCGCCTGAGCCACCTTCCCGCC TGGACACAGGCTGTGTCACTGGAGGTGCTGGACCTGCGAAACAACAGCTTCAGCCTCCTGCCAGGCAG TGCCATGGGTGGCCTGGAGACCAGCCTCCGGCGCCTCTACCTGCAGGGGAATCCACTCAGCTGCTGCG GCAATGGCTGGCTGGCAGCCCAGCTGCACCAGGGCCGTGTGGACGTGGACGCCACCCAGGACCTGAT CTGCCGCTTCAGCTCCCAGGAGGAGGTGTCCCTGAGCCACGTGCGTCCCGAGGACTGTGAGAAGGGG GGACTGAAGAACATCAACCTCATCATCATCCTCACCTTCATACTGGTCTCTGCCATCCTCCTCACCACGCT GGCCGCCTGCTGCTGCGTCCGCCGGCA GAAGTTTAACCAACAGTATAAAGCCTAAAGAAGCCGGGAGA CACTCTAGGTCAGTGGGGGAGCCTGAGGTACAGAGAAGAGTGAGGACTGACTCAAGGTCACACAGTG ATCCGGGATCCCAGAACTCTGGTCTCCAAATTACAGCCCAGGACACCTTTCTCTGCCGCCTGCTGCATCA GTGGGTGACCCCCTTCCCGGGCTGCACTTTGGGTCCAGCTGTGGAAGCCAGAAGTTGGGCGGTTTCAG GGACAGCCGAGAATAATGTTGACCTGTCAGATCAACAAATCTTCACTGAGCATGTATTTTGTGCCACAC CCTGCTCTGGGCACTGGGAATGCTGGGAAATGAGATACATTCCCGCCCTCAAGAATCTCCCAGTCTGGT AGGAGAGAGTGCTGCAGAGCCACGTGGCCGCCACGCAGTGTGCTTAGGGCCTGAGGTGTGAAAGCCC AGGGCTCCAGAGCTCGGCAGGCCCCGCTGGTTTGGTGCGGTGAGTCCTGCCCCGGCTGTGCCAGGGT GAGGGAGGGCCAAGCCCAGGAGGATTTGTCTGAGACATTTCCAAGCAGACTGTTTGTCACGTCTTCTG AGAATGACTTTCAGTCTCTCTGAAAATGAAAAGCTTAGGACCAGAAGAGAGAATTGGAGCTGTACGAG TGTGTCTCGGATCTGGTGTTGTTAGGTGGGCCACGGCGGCTCCAGCAGGGTCTGGTTAAGGGGTCCAG CCCAGCACTGGACCATTCCGTCTCCTGCTCTGGACTTGCCCTCTCCCTTCCTGGCACTCTCATGTTGCATA CCCTGACCCCAGTGCTGCTCTAAGCACCGTCCCTGCCCAGCCCCACTTCTCCATCGCAGCCCCACCTTGG CTGCTGAGCCAGGAGCTAAAACCTTAGATATCTGGTTCTGTTTTGCACCCAGCTTGGCAGATGTGGATT TGAATCCAAGCCTTGTGTCTGCCCCTATGTGACAGCTCTATATTTTATCCCCGT TTTATAAAAGAGGAAA CTGAAGTTCTGAAAATCTCCTTCCAGGGCCCCAGCTAACTAATGCCATAGGTGAGATTCAAACCTTCATC CTTCTGTCTCCAGGGCCTGATCTTTACCACTGCAGGGGCTGCAGGCCGTTAAGTGGACAGGAAGTGGC CCCACATAGCCCGAGCAGGGTCTGGAAGCATCCTGTGCTGTGCACACCTGCTCTCTCCTCTCTCCCAGG CAGGCAGCTGCAGGCGCTCTCCTCCTTCTCTGCCTGTTTCCCTCCTCCCTTCCTTTCCACCCTGGTGTGGG TTCTCCTGTTCTCTCTGTGCTCTTGCATTCTCTCATTCCCTTTTCCTCTATTGAGCAGAGCCTGGAGTTTGA GACTATGGAATCCAACCTCCCCATTGCACAGATGGGGAAACTGAGGCTTAGGAAGAGAATGAAACTTG TGGAGAGCTTATACAGAGCCTCTGGGGGAAAAAAGAGCCCTTATTTGTGGGGTGAGATTGGGGGTTG GACCAGAGTGATGTCCTCTCTCAGCTATCACATCACAAGATAATGCTGGCTCCAAACTTCCTTTCTGTGC CTCATCATGCAAGGATCTTTTTTCCCTCTTACAAAAACAGGTAAAAAGCCTCACCCAGATGACCCCCATC CCTCATACCATGGAGTCATGAGCTGTCTGGGAAGAATGGACGTGCTGGGACCAACTCAAGACCTTGTTT TGCTGTCTTCATCATCTTACCTGTGCTTGGCCCACAGTCTGGCTCATGATGTGGGCTCAGTAATGTGCGA GAAAGTGAAAATGCCACTCTCTCCACCCCATTTTACAGAGGAGAACACCAAGGCCCAGAGGAAGTTAA GGGAGAGTCAATGGGCAGAGCCAGGGCTAGGCCCTGGTGGTGTGTGGAGCACCCAGGCAGACCCAG TCCTGGTTGGGATCACACCCACGGGTGCTACTGCACGTAACACTCCTCCTTAGGCCTGGAGGCCAAGGT GT GGGTCCCCACGCCTGATCTTTGAAAACACTACACAGGGCTGCTGTCACTTCCCAGGGCCCAGGCCTC AGCCCAGGCCTCGGGACCAACTCrTTGWAACCTACCTGAATGTATTAAAAACTAATTTTGGAGAAGC A

SEQ ID NO: 2

MATALSAGGLGPLPRVTSLDLSGNSLYSGLLERLLGEAPSLHTLSLAENSLTRLTRHTF RDMPALEQLDLHSNVLMDIEDGAFEGLPRLTHLNLSRNSLTCISDFSLQQLRVLDLSC NSIEAFQTASQPQAEFQLTWLDLRENKLLHFPDLAALPRLIYLNLSNNLIRLPTGPPQD SKGIHAPSEGWSALPLSAPSGNASGRPLSQLLNLDLSYNEIELIPDSFLEHLTSLCFLNL SRNCLRTFEARRLGSLPCLMLLDLSHNALETLELGARALGSLRTLLLQGNALRDLPPY TFANLASLQRLNLQGNRVSPCGGPDEPGPSGCVAFSGITSLRSLSLVDNEIELLRAGA FLHTPLTELDLSSNPGLEVATGALGGLEASLEVLALQGNGLMVLQVDLPCFICLKRLNL AENRLSHLPAWTQAVSLEVLDLRNNSFSLLPGSAMGGLETSLRRLYLQGNPLSCCGN GWLAAQLHQGRVDVDATQDLICRFSSQEEVSLSHVRPEDCEKGGLKNINLIIILTFILVS AILLTTLAACCCVRRQKFNQQYKA

In a preferred embodiment of this aspect of the invention, the treatment is chemotherapy and / or radiotherapy. Preferably the chemotherapy treatment is selected from a topoisomerase inhibitor, an antitumor antibiotic, or any combination thereof. More preferably the topoisomerase inhibitor is selected from Topotecan, Irinotecan (CPT-11), Etoposide (VP-16), Teniposide, or Mitoxantrone. In another preferred embodiment the antitumor antibiotic is selected from the list consisting of: Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Actinomycin D, Bleomycin, Mitomycin C, Mitoxantrone, or any of their combinations. In a particular relationship, the chemotherapy treatment is etoposide and / or doxorubicin.

In another preferred embodiment of this aspect of the invention the cancer is selected from the list consisting of: leukemia, sarcoma, bladder, breast, brain, colon, stomach, lung, ovarian, thyroid, multiple myeloma cancer. Most preferably the cancer is sarcoma. Even more preferably the sarcoma is selected from the list consisting of chondrosarcoma, osteosarcoma, pleomorphic sarcoma, and synovial sarcoma. Much more preferably it is osteosarcoma. In another particular embodiment, it is Ewing's sarcoma.

METHODS FOR PREDICTING OR FORECASTING THE RESPONSE TO

CANCER TREATMENT OF THE INVENTION

A second aspect of the invention refers to an in vitro method for obtaining useful data to predict or predict the response to cancer treatment in an individual, hereinafter the first method of the invention, which comprises measuring in an isolated biological sample the expression product of GARP. More preferably, the first method of the invention further comprises comparing the amounts obtained with a reference amount.

A third aspect of the invention refers to an in vitro method for predicting or predicting the response to cancer treatment in an individual, hereinafter the second method of the invention, which comprises measuring the expression product of GARP, compare the quantities obtained with a reference quantity, and include the individual in the group of individuals with a lower probability of response to treatment, when the expression of GARP is significantly higher (p <0.05) than that of the reference sample .

There are also in vivo tests. In a series of 11 sarcoma patients for which response data to different first-line treatments are available (A), the authors of the present invention found that the only two complete responses occurred in 2 of the 4 patients with low levels of GARP. On the other hand, the only case in which treatment had no effect on tumor growth (PD) corresponds to a patient with high levels of GARP. Despite the small number of patients included in this analysis, we found significant differences (p = 0.039) between patients with high and low levels of GARP when doing a dichotomous analysis of the complete responses versus the rest of the response options (partial response, stable disease and progressive disease) (B). Therefore, these data suggest that sarcomas expressing high levels of GARP respond worse to antitumor treatments.

In immunohistochemical studies, GARP expression was assessed using a semi-quantitative system in which the following values are assigned depending on the percentage of cells expressing GARP: 1: 0%; 2: <10%; 3: 10-50%; and 4:> 50%. According to this system, the tumors of the patients who responded worse (disease progressive stable disease partial response) to the treatments show GARP expression values 3.6 times higher than the tumors of the patients who presented a complete response.

Therefore, preferably, the individual is included in the group of individuals with a lower probability of response to treatment, when the GARP expression product is at least 1.5 times, more preferably at least 2 times. more preferably at least 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, or 3.5 times the expression product of the reference sample. In a particular embodiment, the individual is included in the group of individuals with a lower probability of response to treatment, when the GARP expression product is at least 3.6 times the expression product of the reference sample.

In a preferred embodiment, the steps of the methods described above can be fully or partially automated and / or computerized, for example, by means of a robotic liquid dispensing equipment, a robotic sensor equipment for the detection of the amount or product of expression, or computerized comparison.

The described comparison of the methods of the invention can be performed manually or computer-aided.

The term "expression product", also called "gene product" refers to the biochemical material, either RNA (eg microRNA) or protein, resulting from the expression of a gene. Sometimes a measure of the amount of gene product is used to infer how active a gene is.

The expression of a biomarker of the invention can be evaluated by any of a wide variety of methods well known to those skilled in the art to detect the expression of a transcribed molecule or its corresponding protein. If the biomarkers are nucleic acid molecules, expression can be detected and / or quantified using nucleic acid hybridization methods, nucleic acid reverse transcription methods, nucleic acid amplification methods, and the like.

Thus, quantification can be performed by polymerase chain reaction (PCR) or a technique that includes PCR, and more preferably by real-time PCT (Q-RT-PCR).

The measurement of the expression levels of a gene, preferably quantitatively, is based on a signal that is obtained directly from the transcripts of said genes (mRNA, microRNA, etc.), and that is directly correlated with the number of molecules of RNA produced by genes. Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said products. On the other hand, it could be carried out by indirect measurement that refers to the measurement obtained from a secondary component or a biological measurement system (for example, the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).

Thus, in a preferred embodiment, the expression of a marker gene is assessed by preparing mRNA / cDNA (i.e., a transcribed polynucleotide) from a sample, and by hybridizing the mRNA / cDNA with a reference polynucleotide that is complementary to a polynucleotide comprising the marker gene, and / or fragments thereof. The cDNA can optionally be amplified using any of several polymerase chain reaction methods prior to hybridization to the reference polynucleotide.

Preferably, the determination of the levels of the GARP expression product is done by immunoassay or immunohistochemistry. More preferably the determination of GARP levels is carried out using a technique that is selected from: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or chip protein. In an even more preferred embodiment, the determination of the GARP expression product is performed by immunohistochemistry.

In a preferred embodiment of this aspect of the invention, the treatment is chemotherapy and / or radiotherapy. Preferably the chemotherapeutic treatment is selected from a topoisomerase inhibitor, an antitumor antibiotic, or any combination thereof. More preferably the topoisomerase inhibitor is selected from Topotecan, Irinotecan (CPT-11), Etoposide (VP-16), Teniposide, or Mitoxantrone. In another preferred embodiment the antitumor antibiotic is selected from the list consisting of: Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Actinomycin D, Bleomycin, Mitomycin C, Mitoxantrone, or any of their combinations. In a particular relationship, the chemotherapy treatment is etoposide and / or doxorubicin.

In another preferred embodiment of this aspect of the invention the cancer is selected from the list consisting of: leukemia, sarcoma, bladder, breast, brain, colon, stomach, lung, ovarian, thyroid, multiple myeloma cancer. Most preferably the cancer is sarcoma. Even more preferably the sarcoma is selected from the list consisting of chondrosarcoma, osteosarcoma, pleomorphic sarcoma, and synovial sarcoma. Much more preferably it is osteosarcoma. In another particular embodiment, it is Ewing's sarcoma.

A "prognostic method" (or "prognostic method") refers to a procedure where, based on clinical analysis, the disease or condition, such as cancer, preferably a sarcoma, that the person suffers is identified and, based on Experiences with other patients suffering from the same disease or illness, such as cancer, preferably sarcoma, predict how the individual's health status will be in the future, that is, the evolution of the disease or illness over time. The term "prognosis" in the present invention also refers to the ability to detect asymptomatic patients or subjects who have a high probability of suffering from a disease or ailment, such as cancer, preferably a sarcoma, even when at the time of diagnosis they do not present said pathologies or obvious manifestations thereof. As those skilled in the art will understand, such an assessment, although preferred, may not normally be correct for 100% of the subjects to be predicted. The term, however, requires that a statistically significant part of the subjects can be identified as having the disease or as having a predisposition to it. Whether a part is statistically significant can be determined out of hand by the person skilled in the art using various well-known statistical evaluation tools, for example, determination of confidence intervals, determination of p-values, Student's t -test, Mann-Whitney test. , etc. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-values are preferably but not limited to 0.2, 0.1,0.05.

The degree of identity existing between two sequences can be determined by conventional methods, for example, by means of standard sequence alignment algorithms that are known in the state of the art, such as BLAST (Altschul SF et al. Basic local alignment search tool. J Mol Biol. 1990 Oct 5; 215 (3): 403-10).

A "biological sample", as defined herein, is a small part of a subject, representative of the whole, and may consist of a biopsy or a sample of body fluid. Biopsies are small pieces of tissue and can be fresh, frozen, or fixed, such as formalin-fixed and paraffin-embedded FFPE. Body fluid samples can be blood, plasma, serum, urine, sputum, cerebrospinal fluid, milk or ductal fluid samples and can also be fresh, frozen or fixed. Samples can be removed surgically, by extraction ie by hypodermic or other needles, by microdissection or laser capture. The sample must contain any suitable biological material to detect the desired biomarker or biomarkers, therefore, said sample must advantageously comprise material from the cells of the subject.

A "tissue sample", "tissue sample" or "isolated tissue / tissue sample " includes, but is not limited to, cells and / or tissues from a subject, obtained by any method known to one of ordinary skill in the art. The tissue sample can be, for example, but not limited to, a biopsy or a fine needle aspirate. Preferably the cells are tumor cells.

In another preferred embodiment, the sample is a body fluid sample such as a plasma, serum and / or blood sample.

The terms "individual", "patient" or "subject" are used interchangeably in the present description, and are not intended to be limiting in any respect, the "individual", "patient" or "subject" may be of any age, sex and physical condition.

As described above, the measurement of quantity or concentration, preferably semi-quantitatively or quantitatively, can be carried out directly or indirectly, as indicated above in the present description. Direct measurement refers to the measurement of the amount or concentration of the expression products, based on a signal that is obtained directly from the expression products, and that is correlated with the number of molecules of said expression products. Said signal - which we can also refer to as an intensity signal - can be obtained, for example, by measuring an intensity value of a chemical or physical property of said expression products. Indirect measurement includes the measurement obtained from a secondary component or a biological measurement system (eg the measurement of cellular responses, ligands, "tags" or enzymatic reaction products).

A "reference sample" refers to a similar sample obtained from a control subject or individual (patient) who does not have the disease or condition, such as a cancer, preferably a sarcoma, that is intended to be diagnosed or prognostic. The "reference quantity" or "reference level" refers to the absolute quantity (or levels) obtained from the reference sample. A "reference sample" can also refer to a similar sample obtained from the same subject or individual at an earlier point in time.

A "reference value" refers to the set of values that are used to interpret the results of diagnostic, prognostic and / or follow-up tests in a subject or individual (patient). For example, the baseline values for a particular test are based on the results of that same test in 95% of the healthy population. The reference values of a test may be different in different groups of people (for example, between women and men, or between children and adults). It may also be referred to as 'reference interval', 'reference limit', and 'normal limit'.

In the context of the present invention, "Response" refers to the clinical outcome of the subject, "Response" can be expressed as overall survival or progression-free survival. Survival of cancer patients is generally adequately expressed by Kaplan-Meier curves, named after Edward L. Kaplan and Paul Meier, who first described it (Kaplan, Meier: J. Amer. Statist. Assn. 53: 457-481). The Kaplan-Meier estimator is also known as the product limit estimator. It is used to estimate the survival function from lifetime data. A graph of the Kaplan-Meier estimate of the survival function is a series of horizontal steps of decreasing magnitude that, when a large enough sample is taken, approximates the true survival function for that population. The value of the survival function between successive distinct sampled observations is assumed to be constant. With respect to the present invention, the Kaplan-Meier estimator can be used to measure the fraction of patients who live for a certain period of time after the start of chemotherapy and / or radiotherapy. The predicted clinical outcome may be survival (overall / progression-free) in months / years from the time of sample collection. It can be survival over a certain period from the sample collection, such as six months or more, one year or more, two years or more, three years or more, four years or more, five years or more, six years or more. In each case, "survival" can refer to "overall survival" or "progression-free survival."

Therefore, in one embodiment, the answer is the clinical outcome, which is "overall survival" (OS). "Overall survival" denotes the chances of a patient staying alive for a group of people with cancer. The decisive question is whether the individual is alive or dead at any given time.

KIT AND DEVICES OF THE INVENTION

A fifth aspect of the present invention refers to a kit or device, hereinafter kit or device of the invention, comprising the elements and / or reagents necessary to quantify the GARP expression product.

In another preferred embodiment, the kit or device of the invention comprises primers, probes and / or antibodies capable of quantifying the GARP expression product, and where:

- the primers or primers are polynucleotide sequences of between 10 and 30 base pairs, more preferably between 15 and 25 base pairs, even more preferably between 18 and 22 base pairs, and still much more preferably around 20 base pairs, having an identity of at least 80%, more preferably at least 90%, still more preferably at least 95%, still much more preferably at least 98%, and particularly 100 %, with a fragment of the sequence complementary to SEQ ID NO: 1

- the probes are polynucleotide sequences of between 30 and 1100 base pairs, more preferably between 100 and 1000 base pairs, and even more preferably between 150 and 500 base pairs, exhibiting an identity of at least 80% , more preferably at least 90%, even more preferably at least 95%, still much more preferably at least 98%, and particularly 100%, with a fragment of the sequences complementary to SEQ ID NO: 1,

- the antibodies are capable of specifically binding to a region formed by the amino acid sequence SEQ ID NO: 2.

In another preferred embodiment of this aspect of the invention, the primers or probes are modified, for example, but not limited to, by radioactive or immunological labeling.

In another preferred embodiment of this aspect of the invention, the oligonucleotides have modifications in some of their nucleotides, such as, for example, but not limited to, nucleotides that have some of their atoms with a radioactive isotope, usually 32P or tritium, immunologically labeled nucleotides , as for example with a digoxigenin molecule, and / or immobilized on a membrane. Several possibilities are known in the state of the art.

In another preferred embodiment of this aspect of the invention, the antibody is human, humanized, or synthetic. In another preferred embodiment, the antibody is monoclonal and / or is labeled with a fluorochrome. Preferably, the flurochrome is selected from the list comprising Fluorescein (FITC), Tetramethylrhodamine and derivatives, Phycoerythrin (PE), PerCP, Cy5, Texas, allophycocyanin, or any combination thereof.

The kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc.

On the other hand, the kit can include all the supports and containers necessary for its start-up and optimization. Preferably, the kit further comprises instructions for carrying out the methods of the invention.

In a preferred embodiment of this aspect of the invention, the treatment is chemotherapy and / or radiotherapy. Preferably the chemotherapeutic treatment is selected from a topoisomerase inhibitor, an antitumor antibiotic, or any combination thereof. More preferably the topoisomerase inhibitor is selected from Topotecan, Irinotecan (CPT-11), Etoposide (VP-16), Teniposide, or Mitoxantrone. In another preferred embodiment the antitumor antibiotic is selected from the list consisting of: Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Actinomycin D, Bleomycin, Mitomycin C, Mitoxantrone, or any of their combinations. In a particular relationship, the chemotherapy treatment is etoposide and / or doxorubicin.

In another preferred embodiment of this aspect of the invention the cancer is selected from the list consisting of: leukemia, sarcoma, bladder, breast, brain, colon, stomach, lung, ovarian, thyroid, multiple myeloma cancer. Most preferably the cancer is sarcoma. Even more preferably the sarcoma is selected from the list consisting of chondrosarcoma, osteosarcoma, pleomorphic sarcoma, and synovial sarcoma. Much more preferably it is osteosarcoma. In another particular embodiment, it is Ewing's sarcoma.

A sixth aspect of the invention relates to the use of the kit or device of the invention, the microarray, or the microarray of the invention, to obtain useful data to predict or predict the response to cancer treatment in an individual.

In a preferred embodiment of this aspect of the invention, the treatment is chemotherapy and / or radiotherapy. Preferably the chemotherapeutic treatment is selected from a topoisomerase inhibitor, an antitumor antibiotic, or any combination thereof. More preferably the topoisomerase inhibitor is selected from Topotecan, Irinotecan (CPT-11), Etoposide (VP-16), Teniposide, or Mitoxantrone. In another preferred embodiment the antitumor antibiotic is selected from the list consisting of: Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Actinomycin D, Bleomycin, Mitomycin C, Mitoxantrone, or any of their combinations. In a particular relationship, the chemotherapy treatment is etoposide and / or doxorubicin.

In another preferred embodiment of this aspect of the invention the cancer is selected from the list consisting of: leukemia, sarcoma, bladder, breast, brain, colon, stomach, lung, ovarian, thyroid, multiple myeloma cancer. Most preferably the cancer is sarcoma. Even more preferably the sarcoma is selected from the list consisting of chondrosarcoma, osteosarcoma, pleomorphic sarcoma, and synovial sarcoma. Much more preferably it is osteosarcoma.

AUTOMATED METHOD OF THE INVENTION

A seventh aspect of the invention relates to a computer program comprising program instructions for making a computer carry out the method according to any of the methods of the invention.

In particular, the invention encompasses computer programs arranged on or within a carrier. The carrier can be any entity or device capable of supporting the program. When the program is incorporated into a signal that can be transported directly by a cable or other device or medium, the carrier can be constituted by said cable or other device or medium. As a variant, the carrier could be an integrated circuit in which the program is included, the integrated circuit being adapted to execute, or to be used in the execution of, the corresponding processes.

For example, the programs could be embedded in a storage medium, such as a ROM memory, a CD ROM memory or a semiconductor ROM memory, a USB memory, or a magnetic recording medium, for example, a floppy disk or a disk. Lasted. Alternatively, the programs could be supported on a transmittable carrier signal. For example, it could be an electrical or optical signal that could be carried over electrical or optical cable, by radio, or by any other means.

The invention also extends to computer programs adapted so that any processing means can carry out the methods of the invention. Such programs may be in the form of source code, object code, an intermediate source of code and object code, for example, as in partially compiled form, or in any other form suitable for use in implementing the processes according to the invention. . Computer programs also cover cloud applications based on this procedure.

An eighth aspect of the invention relates to a computer-readable storage medium comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.

A ninth aspect of the invention relates to a transmittable signal comprising program instructions capable of causing a computer to carry out the steps of any of the methods of the invention.

The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).

A nucleic acid or polynucleotide sequence can comprise all five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil) and / or bases other than the five biologically occurring bases. These bases can serve various purposes, for example, to stabilize or destabilize hybridization; to stimulate or inhibit probe degradation; or as attachment points for detectable residues or shielding residues. For example, a polynucleotide of the invention may contain one or more modified, non-standard, derivatized base moieties, including, but not limited to, N6-methyl-adenine, N6-tert-butyl-benzyl-adenine, imidazole, substituted imidazoles , 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil dihydrouracil, beta-D-galactosylkeosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylininosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5'-

methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wybutoxosine, pesudouracil, cheosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 2-thiouracil, 2-thiouracil, 2-thiouracil, 2-thiouracil , 5-methyluracil (i.e., thymine), uracil-5-oxyacetic acid methyl ester, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, and 5- propynyl pyrimidine. Other examples of modified, non-standard, or derivatized base moieties can be found in US Patent Nos. 6,001,611; 5,955,589; 5,844,106; 5,789,562; 5,750,343; 5,728,525; and 5,679,785. In addition, a nucleic acid or polynucleotide sequence can comprise one or more modified sugar moieties including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and a hexose.

The terms "amino acid sequence", "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

EXAMPLES OF THE INVENTION

This is the first study to analyze GARP expression in human sarcoma samples. The inventors found that 100% (8/8) of osteosarcomas and 73% (8/11) of chondrosarcomas expressed high levels of GARP. This is in agreement with the expression of GARP in BM-MSC and the relatively higher expression of GARP in the osteo and chondrosarcoma cancer cell lines shown in the study.

The current treatment for bone sarcoma is surgery and multimodal chemotherapy and, in some cases, radiation therapy when the tumor cannot be removed surgically. In general, doxorubicin, cisplatin, and iphosfamide represent first-line drugs for the treatment of localized bone sarcoma, while cyclophosphamide in combination with etoposide is important for the treatment of recurrent osteosarcoma and Ewing's sarcoma. The combination of enhanced surgical procedures with efficient chemotherapy (CT) and radiation therapy (ET) has substantially increased the survival rate of patients with bone sarcoma. However, during the past 30 years no further improvement has been achieved, and patients with recurrent or metastatic disease still have a <20% chance of long-term survival despite aggressive therapies. One explanation for this lack of progress is the frequent radioresistance exhibited by osteo and chondrosarcoma tumors.

Materials and methods

Animals

NOD scid gamma (NSG) mice were obtained from the internal stock of the Center for Biomedical Research (CIBM, Granada, Spain). All experiments were carried out in accordance with the Institutional Guidelines for the care and use of laboratory animals in research, with the approval of the Animal Research Ethics Committee of the University of Granada (Granada, Spain).

Cell cultures

The G292, T1-73 and SAOS-2 osteosarcoma cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in advanced DMEM (Invitrogen) with 10% FBS (Biowest), 1% Glutamax (Gibco, Thermo Fisher Scientific) and 100 U / ml penicillin / streptomycin (Gibco, Thermo Fisher Scientific). The Ewing RD-ES sarcoma cell line was grown in RPMI-1640 (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS and 100 U / ml penicillin / streptomycin. The primary cell lines OST-3 and OST-4 were generated from tumor samples surgically resected at the Central University Hospital of Asturias (Oviedo, Spain). OST-3 derives from a conventional osteoblastic osteosarcoma resected from a 10-year-old patient and OST-4 corresponds to a dedifferentiated osteosarcoma from a 69-year-old woman. The T-CDS17 chondrosarcoma cell line was previously reported (Rey, V et al., 2019. J Clin Med 8 (4): 455). The above cell lines were grown in high glucose DMEM (Gibco, Thermo Fisher Scientific) with 10% FBS and 100 U / ml penicillin / streptomycin. All cell lines were kept at 37 ° C with partial pressure levels of 21% O2 and 5% CO2. GARP mRNA levels in cell lines were measured by RT-PCR as described in Supplementary Materials and Methods.

Production of lentiviral vectors (LV) and cell transduction.

LV encoding human GARP-specific shRNAs (shARN MISSION® plasmid DNA, RefSeq: SHCLND-NM_005512; Sigma-Aldrich, MO, USA), referred to in the manuscript as GARPKO1 (TRCN0000005218) and GARPKO2 (TRCN0000005219) were used ). A non-mammalian shRNA control plasmid from pLKO.1-MISSION® (Sigma-Aldrich), referred to in the text LV-CTRL, was used as a control. An LV encoding the human codon optimized GARP cDNA, LV-GARP, (Carrillo-Gálvez et al., Presented) was used to overexpress GARP and LV-CEWP was used as a control. LVs were produced by co-transfecting 293T cells with: 1) LV-CTRL / GARPKO1 / GARPKO2 / LV-GARP / LV-CEWP plasmid, 2) packaging plasmid pCMVAR8.91 and 3) envelope plasmid pMD.G as described previously (Carillo-Gálvez AB et al., 2015. Stem Cell. 33 : 183-195). The LVs were subsequently concentrated (x30) using centrifugal filter devices (Amicon Ultra-15, 100 kD, Merck). GARP was silenced in BM-MSC, G292, T1-73, SAOS-2 and OST-4 cells as previously described (Carrillo-Gálvez et al., Stem Cells 2015). To overexpress GARP (GARP ++), RD-ES and SAOS-2 cells were transduced once with concentrated LV-GARP (30x), for 5 hours and subsequently expanded, while G292 cells were transduced twice on consecutive days with LV-GARP (x30). Cells were transduced with LV-CEWP as a control. GARP expression was analyzed by flow cytometry 4 days after transduction as described in Supplementary Materials and Methods.

In vitro cell proliferation analysis.

The proliferation of BM-MSC and tumor cells was analyzed using the Roche xCelligence Real-Time Cell Analyzer System (Roche Applied System, Penzberg, Germany) as previously described (Carillo-Gálvez AB et al., 2015. Stem Cell. 33: 183-195). Plates E were placed on the device station in the incubator (21% O2 / 5% CO2 at 37 ° C) for continuous recording of impedance, as reflected by cell index. In some experiments, the Proliferation was measured as follows: 50,000 cells / well of NT and GARP ++ G292, SAOS-2 and RD-ES cells were placed in a 12-well plate. When the cell cultures reached 80-90% confluence, the cells were harvested, counted, and reseeded at the same concentration. In some cases, 5 hours after cell plating, cells were treated with 10 pM of SB431542 (Sigma-Aldrich) or 2.5 gg / ml of anti-TGF-pi / 2/3 Ab (11D1, R&D Systems, Minneapolis , MN).

In vivo tumor growth analysis.

NT, LV-CTRL, GARPKO2 and GARP ++ G292 cells (4x106) were injected subcutaneously into NSG mice and tumor growth was measured every 6-7 days. Tumor volume was calculated using the formula: (n / 6) x (a2 x b) where a and b are the values for the smallest and largest tumor diameter, respectively. After 2-3 months, mice were sacrificed, tumors were removed, and tumor volumes and weights were measured. Immunohistochemistry was performed on paraffin-embedded tissue sections with monoclonal antibody against human Ki-67 (MIB-1, DAKO) and an anti-Smad3 antibody (phospho S423 S425) (EP823Y, Abcam) as described in Supplementary Materials and Methods .

Cell viability in response to etoposide and doxorubicin

Cell viability was measured using the CellTiter-Blue reagent (Promega) and following the manufacturer's instructions. 10,000 plates / well of NT and GARP ++ G292, SAOS-2 and RD-ES were placed in 96-well plates. 5 hours later, the cells were treated with 10 gM of SB431542 or 2.5 gg / ml of anti-TGF-p 1/2/3 Ab. CellTiter-Blue was added to the RD-ES culture after 24 hours and to the G292 and SA0S-2 cultures after 48 hours. Cells were incubated with the reagent for 4 hours and fluorescence was measured at 560 nm on a Glomax multiple detection system (Promega) following the manufacturer's instructions. This experiment was also performed on cells treated with etoposide and doxorubicin. For this, 10,000 cells / well of NT and GARP ++ G292, SAOS-2 and RD-ES were plated in 96-well plates and 24 hours later 2.5 gM of doxorubicin or different concentrations of etoposide were added to the plates: 2.5 gM, 5 gM, 10 gM and 20 gM for RD-ES and SAOS-2 tumor cells and 10 gM, 20 gM, 40 gM and 80 gM for G292 tumor cells. CellTiter-Blue addition and analysis was performed as explained above. In addition, apoptosis was measured as described in Supplementary Materials and Methods.

Active TGF-p measurement

NT and GARP ++ G292, SAOS-2 and RD-ES cells (40,000 cells / well) were co-cultured with SMAD-binding element (SBE) -HEK293 cells (40,000 cells / well; BPS Bioscience) in assay plates of 96 wells (Sigma-Aldrich) using DMEM supplemented with 0.5% FBS. After 18 hours, SBE activity was analyzed using the One Step Luciferase Assay System (BPS Bioscience) on a Glomax Multiple Detection System (Promega) following the manufacturer's instructions.

Clonogenicity test

The NT, GARP ++ SAOS-2 and RD-ES cells were seeded in 6-well plates at the following densities: 2000, 4000, 8000 and 160000 cells / well (NT) and 1000, 2000, 4000, 8000 cells / well ( GARP ++), and were irradiated with 0, 2, 4 and 8 Gy, respectively, using a dose of 1.66 Gy min-1 in an L. Shepherd & associates MARK-I model 30 gamma irradiator at the Experimental Radiology Unit of the University from Granada (Spain). In some experiments, 10 pM of SB431542 was added 24 hours before irradiation. The cells were subsequently kept in culture until the appearance of colonies that could be counted (7-9 days after irradiation). Cells were fixed and stained with crystal violet and colonies were counted (> 50 cells / colony were considered for survival). The survival fraction was calculated as previously described (Franken NAP, Rodermond HM, et al., 2006, Nature protocols).

H2AX Phosphorylation Analysis

For the detection of double stranded DNA breaks (DSB), phosphorylated H2AX (y-H2AX) was measured by flow cytometry using the H2AX FlowCellect® Histone Phosphorylation Assay Kit (Millipore, MA, USA). To induce DSB, NT and GARP ++ SAOS-2 and RD-ES tumor cells were seeded in 12-well plates (50,000 cells / well), with or without 10 pM SB431542 and the next day exposed to an irradiation dose of 4 Gy. After 2 hours, cells were harvested and stained for Y-H2AX according to the manufacturer's instructions. To analyze the number of Y-H2AX foci / cells, cells were irradiated and stained for Y-H2AX as described above and subsequently acquired on an ImageStream X Mark II imaging flow cytometer (Millipore) and analyzed using the IDEAS software (Spot Wizard).

Patients and tissue samples.

Paraffin embedded tissues of 89 sarcoma patients who underwent resection of their tumors at the Central University Hospital of Asturias (HUCA) were studied. The samples and data of the donors included in this study were provided by the Principality of Asturias Biobank (PT17 / 0015/0023), integrated into the National Network of Biobanks of Spain, and were processed following standard operating procedures with the approval appropriate of the Ethical and Scientific Committees. . All samples of human origin were obtained with the signed informed consent. Sixty percent of the cases were men; the mean age at diagnosis was 49 years (range 2 - 89 years). Twenty-eight (31%) patients had a history of tobacco use (15 current smokers and 13 former smokers). Tumor grade was assessed in H and E stained preparations using the classification system of the French Federation of Comprehensive Cancer Centers (Torin, J et al. 2018. Neoplasia 20: 44-56). The clinicopathological characteristics of the patients are included in Table S1. How the tissue microarray was constructed is detailed in the companion materials and methods.

Immunohistochemistry

The formalin-fixed and paraffin-embedded tissues were cut into 3 µm sections and dried on Flex IHC microscope slides (Dako, Glostrup, Denmark). The sections were deparaffinized with standard xylene and hydrated through graduated alcohols in water. Antigen retrieval was performed using Envision Flex Target Retrieval solution, low pH (Dako, Glostrup, Denmark). Staining was performed at room temperature on an automated staining workstation (Dako Autostainer Plus) with an anti-LRRC32 / GARP antibody (Abcam # ab231214) at a 1: 300 dilution using the Dako EnVision Flex visualization system (Dako Autostainer , Denmark). Hematoxylin counterstaining was the final step. Two independent observers scored immunostaining blinded to clinical data using a semi-quantitative scoring system based on the percentage of stained cells (1: 0%; 2: <10%; 3: 10-50%; and 4:> 50%). and the staining intensity (0: no expression; 1: low intensity; and 2: high intensity). Each sample received a score value resulting from the multiplication of both scores, and the mean value in the resulting distribution was used to discriminate low and high expression samples.

Statistic analysis

For the in vitro experiments and the in vivo tumor growth experiment, statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). All data are represented as the mean (SD) of at least three independent experiments unless otherwise indicated in the figure legend. Multiple comparisons of the data were made using the one-way ANOVA, followed by the Bonferroni post test. A value of p <0.05 was considered significant. The experimental results distributed among the different clinical groups of tumors were evaluated for their importance using the x2 test (with Yates correction, when appropriate). For statistical purposes, the continuous variables (age, tumor size) were dichotomized, taking the mean value as the cut-off point. Survival curves were calculated using the Kaplan-Meier product limit estimate. Differences between survival times were analyzed using the log-rank method and the Hazard Ratio was calculated using a univariate Cox regression analysis. Multivariate Cox proportional hazards models (advanced Wald method) were used to examine the relative impact of these statistically significant variables in the univariate analysis. All statistical analyzes were carried out with the SPSS 24 software package (SPSS, IBM corp.). All tests were two-tailed and p values <0.05 were considered statistically significant.

Table S1.

Table S1. Distribution of sarcoma cases (n = 89) according to their GARP expression level across categories of the indicated patient characteristics and tumor dinicopathologic parameters. P values are shown.

Table S2.

Table S2. Univariate and multivariate Cox analysis.

RESULTS

GARP is expressed in several bone sarcoma cancer cell lines and its silencing blocks its proliferation

An analysis of the Cancer Cell Lines Encyclopedia (CCLE) database revealed that GARP mRNA expression was found predominantly in several sarcoma cancer cell lines, including giant cell tumors, osteosarcoma, and chondrosarcoma, but also in Hodgkin lymphoma and B-cell cell lines (Figure 1A). We selected three osteosarcoma cell lines with varying levels of GARP (G292, T1-73 and SAOS-2), one low GARP Ewing sarcoma cell line (RD-ES), two glioblastoma cell lines (U87, U251) and two carcinoma cell lines (HT-29, MCF-7) and compared their expression of GARP with MSCs derived from human bone marrow (BM). Our data showed that osteosarcoma cell lines G292 and T1-73 expressed similar levels of GARP compared to primary BM-MSCs, while glioma and carcinoma cell lines expressed relatively low levels of GARP mRNA (Figure 1B). We also analyzed the expression of surface GARP by flow cytometry that corroborated the relative levels of GARP mRNA expression (G292>BM-MSC>T1-73>SAOS-2> RD-ES) (Figure 1C). Importantly, as previously demonstrated in human ASCs [25], we found that GARP silencing (Supplemental Fig. 1) in BM-MSC, using lentiviral vectors encoding two GARP-specific shRNAs (GARPKO1 and GARPKO2), decreased substantially its proliferative capacity compared to non-transduced (NT) and control transduced (LV-CTRL) BM-MSCs (Figure 1D). Similarly, GARP silencing in G292, T1-73, and SAOS-2 cell lines reduced their proliferative capacity compared to NT and LV-CTRL cells (Figure 1D and E) and resulted in increased death. cell by apoptosis (Figure 1F).

GARP promotes the proliferation of bone sarcoma cell lines through the activation of TGF-p.

Next, we wanted to analyze the effect of GARP overexpression on the proliferation of bone sarcoma cells. To this end, we overexpress GARP (GARP ++) in the Ewing sarcoma cell lines G292, SAOS-2, and RD-ES using an LV encoding a codon-optimized human GARP cDNA under the control of the CMV promoter (Figure 2A). In parallel, the control cells were transduced with an LV encoding the enhanced green fluorescent protein (EGFP +). GARP overexpression significantly increased the proliferative capacity of all cell lines compared to NT and EGFP + cells (Figure 2B and C). Since TGF-p is a growth factor for osteosarcoma cells [28] and GARP has been shown to promote TGF-p activation [29], we evaluated whether GARP overexpression could increase TGF-p activation. To this end, we co-cultured NT and GARP ++ bone sarcoma cells with a SMAD-binding element (SBE) -HEK293 TGF-p indicator cell line. We found that GARP overexpression significantly increased TGF-p activation by G292, SAOS-2 and RD-ES cells (Figure 2D). Blocking TGF-p signaling with SB431542 or neutralizing TGF-p using an anti-TGF-p1 / 2/3 antibody, significantly reduced the proliferation of GARP ++ cells (Figure 2F). In summary, these data show that GARP promotes the proliferation of bone sarcoma cell lines by promoting the activation of TGF-p.

GARP increases the growth of G292 tumors in vivo

To analyze whether GARP expression levels are also important for tumor growth in vivo, we injected NT, GARP ++, LV-CTRL and GARPKO2 G292 cells subcutaneously into NSG mice and followed tumor growth over time. Consistent with in vitro data, GARP ++ G292 exhibited significantly greater in vivo growth compared to NT G292, while smaller tumors were found in only 3 of 8 mice injected with GARPKO2 G292 cells (Figure 3A, B, and C). Furthermore, both volumes and weights of GARP ++ tumors isolated from sacrificed mice were significantly increased compared to NT tumors (Figure 3B). In contrast, the weights and volumes of GARPKO tumors were significantly lower compared to LV-CTRL tumors (Figure 3C). We performed Ki67 immunohistochemistry to analyze the cell proliferation rate in the different tumors and found that GARP ++ tumors contained a significantly higher percentage of Ki67 + cells compared to NT tumors. An opposite trend was observed between GARPKO and LV-CTRL tumors, but the difference was not statistically significant. This could be due to the fact that only three NSG mice injected with GARPKO G292 cells showed the presence of a tumor, possibly with a higher proliferative capacity. Finally, we analyzed the activation of SMAD3 in GARP ++ and NT tumors by immunohistochemistry and observed a higher fraction of cells with high SMAD3 phosphorylation in GARP ++ tumors compared to NT tumors (Figure 3F). In summary, our data show that the modulation of GARP expression in G292 has profound effects on its growth in vivo, corroborating our in vitro data.

Bone sarcoma cell lines that overexpress GARP are more resistant to TGF-p-dependent etoposide and doxorubicin-induced cell death

Previous studies on GARP in the context of cancer have focused on the inhibition of the immune response through increased activation of TGF-p, especially the induction of foxp3 Tregs [18-20]. However, no studies have analyzed the effect of GARP expression on resistance to chemotherapeutic drugs or irradiation. To this end, we added different concentrations of the DNA damaging agent etoposide to the NT, EGFP and GARP ++ G292, SAOS-2 and RD-ES cell lines and analyzed cell survival. (CelltiterBIue) and apoptosis (7AAD / Annexin V) after 24 hours. We found that GARP overexpression increased cell survival (Figure 4A) and reduced apoptosis (Supplementary Figure S2) of all tumor cell lines in response to etoposide. Inhibition of TGF-P signaling (SB431542) or neutralization of TGF-P (1D11) significantly reversed the survival of GARP-overexpressing cells, suggesting a TGF-P-dependent mechanism (Figure 4B). Similarly, GARP ++ cells were more resistant to doxorubicin-induced cell death, although to a lesser extent compared to etoposide. Again, inhibition of TGF-P signaling reversed the protective effect conferred by GARP (Figure 4C).

Sarcoma cells that overexpress GARP show reduced sensitivity to radiation-induced cell death and DNA damage

We then performed a clonogenic assay to assess the relative resistance of NT and GARP ++ sarcoma cells to radiation-induced cell death. Pilot experiments showed that G292 did not form countable colonies, therefore, experiments were performed only with SAOS-2 and RD-ES cell lines. The SAOS-2 and RD-ES NT and GARP ++ cell lines were subjected to different doses of irradiation (2, 4 and 8 Gy), in the presence or absence of SB431542, and the colony formation capacity was followed for approximately two weeks. We found that GARP ++ SAOS-2 and RD-ES cells were significantly more resistant to irradiation-induced cell death compared to NT cells. Inhibition of TGF-P signaling reduced the radioresistance conferred by GARP in both cell lines (Figure 5A and B). TGF-P has been shown to protect cells against radiation-induced DNA damage through its effect on DNA repair [30]. Therefore, we irradiated NT and GARP ++ SAOS-2 and RD-ES cells, cultured with or without SB431542 for 24 hours, with 4 Gy and analyzed the induction of DNA damage as visualized by the phosphorylation of H2AX before and two hours after irradiation. We found that irradiation induced significantly lower levels of y-H2AX in GARP ++ cells compared to NT cells. Interestingly, we found that blocking TGF-P signaling increased the amount of DNA damage in GARP ++ SAOS-2 and RD-ES cells to that of NT cells, implying that TGF-P is resistant to damage. of DNA induced by irradiation (Figure 5C and RE). Finally, to better visualize DNA damage in the nuclei, we repeated the irradiation of GARP ++ and NT SAOS-2 and RD-ES cells, as described above, and analyzed H2AX activation using an Imagestream flow cytometer. What Earlier, we observed that GARP ++ cells were more resistant to irradiation-induced DNA damage, which was dependent on TGF-p signaling (Figure 5E and F). In summary, these data show that GARP, through its activation of TGF-p, can protect tumor cells against radiation-induced cell death and DNA damage, suggesting that GARP could represent a therapeutic target and possibly a predictive biomarker of chemo / radioresistance of bone sarcomas.

GARP is highly expressed in human osteo, chondro, and pleiomorphic sarcomas and is associated with poor survival / prognosis.

The clinical relevance of GARP expression in sarcomas is unknown. Therefore, we studied GARP expression in primary human bone sarcomas and its correlation with clinical data. We first analyzed the expression of GARP in cell lines derived from primary patients with two osteosarcomas (OST-3, OST-4) and one chondrosarcoma (T-CDS17) [26]. Interestingly, we found that all cell lines expressed GARP, albeit at variable levels (Figure 6A). The OST-4 cell lines expressed GARP at a high level and the silencing of GARP in this cell line reduced its proliferative capacity, similar to what we observed using the primary cell lines BM-MSC and bone sarcoma cancer (Figure 6B).

We then set out to analyze GARP expression in a collection of tissue microarrays, including samples from 89 patients with 10 types of sarcomas, by immunohistochemistry. We found that GARP was expressed at high levels in 43 (48%) of the sarcoma samples, including all cases of osteosarcoma and undifferentiated pleomorphic sarcoma (Figure 7A and B). None of the clinicopathological variables analyzed showed a significant correlation with the expression level of GARP (Table S1), although a trend towards a higher grade (p = 0.15) and mitotic count score (P = 0.068) was observed in tumors showing high expression of GARP (Figure 7B). Interestingly, the cases that expressed low levels of GARP had a significantly longer survival time than those with high expression (94 months (CI 87-100) vs 41 months (CI 28-54), respectively; HR 19; p = 0 , 0001). The 5-year survival rate was 95.5% for low-expression cases and 35.3% for high-expression cases (Fig. 7C). Tumor grade, tumor type, mitotic count, necrosis, and GARP expression level were included in the multivariate survival analysis. Tumor necrosis showed an independent prognostic value (p = 0.024, HR 2.643). It is important It should be noted that a trend was observed towards the expression of GARP that has an independent prognostic value (p = 0.056) (Table S2).

Finally, in a series of 11 sarcoma patients where we have the responses to various first-line treatments (Figure 8A), we observed that the only two complete responders (CR) occurred in two out of four patients with low GARP levels. On the other hand, the only case in which the treatment had no effect corresponded to a patient with high levels of GARP. Despite the low number of patients, we detected a significant difference (p = 0.039) between patients with high and low GARP expression who performed a dichotomous analysis of the complete response versus the other response options (partial response, stable disease and progressive disease (Figure 8B) Therefore, these results suggest that sarcomas expressing high levels of GARP have poorer responses to antitumor treatments.

Claims (22)

1 GARP for use as a biomarker to predict or predict response to cancer treatment in an individual. 2. - An in vitro method for obtaining useful data to predict or predict the response to cancer treatment in an individual that comprises measuring the GARP expression product in an isolated biological sample. 3. - The in vitro method of obtaining useful data to predict or predict the response to cancer treatment in an individual according to the preceding claim, which further comprises comparing the quantities obtained with a reference quantity. 4. - An in vitro method to predict or predict the response to cancer treatment in an individual that comprises measuring the GARP expression product in an isolated biological sample, comparing the quantities obtained with a reference quantity, and including the individual in the group of individuals with a lower probability of response to treatment, when the GARP expression product is significantly higher (p <0.05) than that of the reference sample. 5. - An in vitro method to predict or predict the response to cancer treatment in an individual according to the preceding claim, where the individual is included in the group of individuals with a lower probability of response to treatment, when the expression product of GARP is at least 1.3 times 1.4; 1.5; 1.6; 2 times, 2.5 times, 3 times, or at least 3.5 times the expression of the reference sample. 6. - The method according to any of claims 1-5, wherein the determination of the expression product of GARP is carried out by means of an immunoassay or immunohistochemistry. 7. - The method according to any of claims 2-6, where the determination of GARP levels is performed by a technique selected from: immunoblot, enzyme-linked immunosorbent assay (ELISA), linear immunoassay (LIA), radioimmunoassay (RIA), immunofluorescence, x-map or protein chips. 8. - The method according to the preceding claim, where the determination of the GARP expression product is carried out by immunohistochemistry. 9. - The GARP biomarker for use according to claim 1, or the method according to any of claims 2-8, where the treatment is chemotherapy (etopoxide and doxorubicin) and / or radiotherapy. 10. - The method according to any of the preceding claims, wherein the cancer is selected from the list consisting of: leukemia, sarcoma, bladder, breast, brain, colon, stomach, lung, ovarian, thyroid, multiple myeloma. 11. - The method according to any of the preceding claims, wherein the cancer is sarcoma. 12. - The method according to the preceding claim, wherein the sarcoma is selected from: chondrosarcoma, osteosarcoma, pleomorphic sarcoma and synovial sarcoma. 13. - The methods according to the preceding claim, wherein the sarcoma is osteosarcoma or Ewing's sarcoma. 14. - A kit or device, comprising the elements and / or reagents necessary to quantify the levels of GARP. 15. - The kit or device according to the preceding claim, which comprises primers, probes and / or antibodies capable of quantifying the GARP expression product, and where: - the primers or primers are polynucleotide sequences of between 10 and 30 base pairs, more preferably between 15 and 25 base pairs, even more preferably between 18 and 22 base pairs, and still much more preferably around 20 base pairs, having an identity of at least 80%, more preferably at least 90%, still more preferably at least 95%, still much more preferably at least 98%, and particularly 100 %, with a fragment of the sequence complementary to SEQ ID NO: 1 - the probes are polynucleotide sequences of between 30 and 1100 base pairs, more preferably between 100 and 1000 base pairs, and even more preferably between 150 and 500 base pairs, exhibiting an identity of at least 80% , more preferably at least 90%, even more preferably at least 95%, still much more preferably at least 98%, and particularly 100%, with a fragment of the sequences complementary to SEQ ID NO: 1, - the antibodies are capable of specifically binding to a region formed by the amino acid sequence SEQ ID NO: 2. 16. The kit or device according to any of claims 14-15, wherein the antibody has the sequence SEQ ID NO: 2 SEQ ID NO: 3 QELPPYTFASLASLQRLNLQGNQVSPCGGPAEPGPPGCVDFSGIPTLHVL NMAGNSMGMLRAGSFLHTPLTELDLSTNPGLDVATGALVGLEASLEVLEL QGNGLTVLRVDLPCFLRLKRLNLAENQLSHLPAWTRAVSLEVLDLRNNSF SLLPGNAMGGLETSLRRLYLQGNPLSCCGNGWLAAQLHQGRVDVDATQDL ICRFGSQEELSLSLVRPEDCEKGGLKNVNLILLLSFTLVSAIVL. 16. The kit or device according to any of claims 14-15, further comprising: a) a solid support that has attached a primary antibody b) a solution of the detection antibody, labeled with an enzymatic marker; and c) a reagent. 17-. The use of a kit according to any of claims 14-16 to predict or predict response to cancer treatment. 18. - The use of a kit according to claim 17, where the cancer is selected from the list consisting of: leukemia, sarcoma, bladder, breast, brain, colon, stomach, lung, ovarian, thyroid, multiple myeloma . 19. The use of a kit according to any of claims 17-18, wherein the cancer is sarcoma. 20. - The use of a kit according to the preceding claim, where the sarcoma is selected from: chondrosarcoma, osteosarcoma, pleomorphic sarcoma and synovial sarcoma. 21. - The use of a kit according to any of claims 11-12, wherein the sarcoma is osteosarcoma. 22. The use of a kit according to any of claims 11-12, wherein the sarcoma is Ewing's sarcoma.