EP3213082A1 - Vav3 as a marker for cancer - Google Patents

Vav3 as a marker for cancer

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Publication number
EP3213082A1
EP3213082A1 EP15785109.8A EP15785109A EP3213082A1 EP 3213082 A1 EP3213082 A1 EP 3213082A1 EP 15785109 A EP15785109 A EP 15785109A EP 3213082 A1 EP3213082 A1 EP 3213082A1
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EP
European Patent Office
Prior art keywords
cancer
vav3var2
grade
stage
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15785109.8A
Other languages
German (de)
French (fr)
Inventor
Maximilian BÖSCH
Alain Gustave ZEIMET
Daniel Uwe REIMER
Sieghart SOPPER
Dominik Wolf
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oncotyrol Center for Personalized Cancer Medicine GmbH
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Oncotyrol Center for Personalized Cancer Medicine GmbH
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Publication of EP3213082A1 publication Critical patent/EP3213082A1/en
Withdrawn legal-status Critical Current

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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
    • G01N33/57449—Specifically defined cancers of ovaries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
    • 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

  • VAV3 as a marker for cancer
  • the present invention relates to the field of biomarkers for diagnosing cancer, especially ovarian cancer or endometrial can ⁇ cer .
  • Cancers are a leading cause of death worldwide, causing more than 8 million deaths in 2012 (WHO fact sheet No. 297 on cancer, as of February 2014) .
  • Cancer is a generic term for a large group of diseases that can affect any part of the body.
  • Other terms used are malignant tumours and neoplasms.
  • One defining feature of cancer is the rapid creation of abnormal cells that grow be ⁇ yond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. This process is referred to as metastasis. Metastases are the major cause of death from cancer.
  • Ovarian cancer is a frequent form of cancer, 239000 inci ⁇ dences and 152000 deaths of women were estimated for the year 2012.
  • epithe ⁇ lial ovarian cancer which results from malignant transformation of the ovarian surface epithelium (Hennessy et al . , 2009) .
  • the lifetime incidence risk is about 1 in 70 (women) .
  • Identifiable genetic predispositions such as BRCA1/BRCA2 mutations are pre ⁇ sent in only 10-15% of patients.
  • Epithelial ovarian cancers are typically classified by histopathological grade (1-3), stages (I-IV) and appearance into serous (most common), mucinous and endometrioid subtypes.
  • Hennessy et al give an overview over the standard of care for epithelial ovarian cancer. If epithelial ovarian cancer is suspected on the basis of physical examination and imaging, an exploratory laparotomy is usually performed for histological confirmation and tumor debulking, i.e. the partial removal of tumor tissue (also known as cytoreduction) . For many patients with early stage disease with well-differentiated tu ⁇ mors (i.e. grade 1), systemic therapy with chemotherapeutic com ⁇ pounds (which has severe side-effects) is not warranted, as 90% of them have long-term disease free survival without chemothera- Prinic treatment.
  • Carboplatin or platinum-based therapies in general
  • paclitaxel a taxane
  • angiogenesis inhibitor bevacizumab has been introduced recently (Perren et al . , 2011) .
  • others have ovarian cancers that only respond insufficiently (e.g.
  • biomarkers that give a clear guidance for what treatment options to choose (i.e. targeted therapy) are even harder to come by.
  • the present invention provides the use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a cancer marker.
  • VAV3 is a guanine nucleotide exchange factor for Rho/Rac GTPases having core functions in cytoskeletal rearrangements and cell migration (Hornstein et al . , 2004) .
  • VAV3 has oncogenic properties in a variety of tumor entities, including leukemia, lymphoma, breast cancer, prostate cancer, and gastric cancer (cf . e.g. Chang et al . , 2012 and Lin et al . , 2012 A) .
  • VAV3 ex ⁇ pression increases during prostate cancer progression, and high expression of VAV3 also correlates with prostate cancer recur- rence (Lin et al . , 2012 B) .
  • VAV3 comprises the transcript variants VAV3varl (NCBI RefSeq entry NM_006113.4 for H. sapiens) and VAV3var2 (NCBI RefSeq entry NM_001079874.1 for H. sapiens), whose expres ⁇ sion has never been separately investigated in a cancer setting.
  • CSC Cancer stem cells
  • a tran ⁇ script or protein that is overexpressed in CSCs may be a suit ⁇ able cancer marker.
  • the relevance of CSCs in cancer is also dis ⁇ cussed in Pattabiraman & Weinberg, 2014.
  • VAV3 (comprising VAV3varl and VAV3var2) was se ⁇ lectively overexpressed in ovarian cancer stem cells recovered from ovarian cancer cell lines.
  • VAV3var2 was overexpressed in ovarian CSCs (Fig. 1) .
  • tumoral VAV3 expression is almost exclusively due to expression of VAV3var2.
  • VAV3 and especially VAV3var2 are a robust pre ⁇ dictor of patient outcome in ovarian cancer, in particular in regard to relapse in general, and/or response to platinum-based chemotherapies, taxane-based chemotherapies and/or anti- angiogenic chemotherapies.
  • VAV3 (especially VAV3var2) being a robust marker for CSCs - as found in the course of the present invention - is hence also a marker for drug-resistance such as insufficient re ⁇ sponse to the chemotherapies mentioned in the paragraph above. This applies especially to anti-angiogenetic inhibitors, as VAV3 has an important function in tumor angiogenesis (cf. e.g. Hunter et al, 2006 and Brantley-Sieders et al . , 2009).
  • VAV3 (but not VAV3var2) is known from the following cancer-related patent applications:
  • the WO 2012/066451 Al relates to prognostic and predictive gene signatures for colon cancer.
  • the WO 2013/052480 Al relates to a marker-based prognostic risk score in colon cancer.
  • the WO 2010/145796 A2 relates to biomarkers and methods for determining efficacy of anti-EGFR antibodies in cancer therapy.
  • the WO 2007/025044 A2 relates to biomarkers and methods for determining sensitivity to epidermal growth factor receptor modulators .
  • the WO 2013/049398 A2 recites VAV3 as one of many biomarkers for cancer.
  • the EP 2 388 336 Al relates to methods and kits for diagnos ⁇ ing colorectal cancer.
  • Paragraph [0045] of the document refers to Table 1 of the document for 2002 RNAs that are indicative of the presence or absence of colorectal cancer.
  • the entry refer ⁇ ring to SEQ ID NO: 108 in the table contains the identifiers "220131_at", “FXYD7 " and "NM_022006.1”, all of which are in fact related to the FXYD domain-containing ion transport regulator 7 (FXYD7) .
  • SEQ ID NO: 108 of the sequence listing of EP 2 388 336 Al contains the VAV3var2 transcript sequence, which is clearly a mistake as FXYD7 is another gene. Consequently, VAV3var2 is not disclosed or suggested as a cancer marker in the document .
  • VAV3.1 major transcript variants of VAV3, one of them being termed "VAV3.1".
  • the length of human VAV3.1 is disclosed to be 3062 bases (p. 145, left column, 2 nd paragraph of the document) , which is shorter than the VAV3var2 transcript (3115 bases, cf . SEQ ID NO: 2) .
  • the divergence point disclosed for VAV3.1 (Fig. 1 of the document) is different from the one of the VAV3var2 transcript.
  • the docu ⁇ ment does not disclose VAV3.1 as a cancer marker for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient or for diagnosing of a cancer in a tissue sample or in a body fluid sample.
  • VAV3.1 is disclosed to be expressed in normal colon and only a few tumor cell lines (Fig. 5 of the document), it is not even disclosed to be expressed in both ovarian cancer cell lines tested.
  • VAV3.1 as disclosed in the document is not VAV3var2
  • the skilled person would hence not draw the conclusion from the document that VAV3.1 is a cancer marker.
  • the document does not anticipate or suggest the pre ⁇ sent invention.
  • VAV3var2 (NM_001079874, called VAV3.1 in Qi et al . ) is an oncogene.
  • the document does not disclose VAV3var2 as a cancer marker for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient or for diagnosing of a cancer in a tissue sample or in a body fluid sample.
  • Qi et al . does not relate to ovarian cancer.
  • Fig. 5C of the document a reduction of transcription of VAV3var2 upon addition of astragaloside IV, a compound asserted to be antipro ⁇ liferative, to HepG2 cells is shown.
  • VAV3var2 is a can ⁇ cer marker for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient or for diagnosing of a cancer in a tissue sample or in a body fluid sample.
  • the present invention is the first to provide the use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a can ⁇ cer marker, said use being for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient, preferably wherein said chemotherapeutic compound is a platinum-based therapeutic compound, preferably cisplatin or carboplatin, or an angiogenic inhibitor, preferably bevacizumab, or a taxane, or combinations thereof.
  • chemotherapeutic compound is a platinum-based therapeutic compound, preferably cisplatin or carboplatin, or an angiogenic inhibitor, preferably bevacizumab, or a taxane, or combinations thereof.
  • a cancer sample can also be obtained by biopsy of tumours of said patient or, in the case of liquid tumours, by obtaining body fluids from said patient.
  • a biopsy is typically a exploratory laparotomy of the tumour tissue (Hennessy et al . , 2009) .
  • the patient has been diag ⁇ nosed or is suspected of having cancer before applying the in ⁇ ventive use of treatment determination.
  • Platinum based compounds are widely used in chemotherapy. They comprise cisplatin, carboplatin, oxaliplatin (US 4,169,846), satraplatin (e.g. WO 2005/077385 A2), picoplatin, nedaplatin and triplatin.
  • Angiogenic inhibitors are also widely used in chemotherapy.
  • the most prominent one is bevacizumab which is an inhibitor of the VEGF pathway (see e.g. WO 1994/010202 Al, WO 1998/045331 A2 ) .
  • Taxanes are widely used in chemotherapy as well. The most prominent taxanes are paclitaxel and docetaxel.
  • Said inventive use is alternatively for diagnosing of a can ⁇ cer in a tissue sample or in a body fluid sample, wherein VAV3var2 protein or VAV3var2 mRNA is used. It is evident that the tissue sample can be a tumor tissue sample or a putative tu ⁇ mor tissue sample (see also Example 1 and Figs. 2-4) .
  • VAV3varl is significantly longer than VAV3var2 on transcript level (4,776 bp versus 3,115 bp) . This size difference is due to significant truncation of VAV3var2 at the 5' -end, whereas 96% of the remaining sequence is identical to VAV3varl .
  • VAV3varl NCBI Protein database entry NP_006104.4 for H. sapiens
  • VAV3var2 NCBI Pro ⁇ tein database entry NP_001073343.1 for H. sapiens
  • VAV3 belonqs to the evolutionary conserved VAV family of qua- nine nucleotide exchanqe factors, which also includes the VAV proteins VAV1 and VAV2.
  • VAV1-VAV3 share sequence homoloqy which may also result in partially overlappinq protein function.
  • VAV1 and VAV2 in contrast to VAV3 did not show differential expression between CSC and non-CSC.
  • the cancer is a leukemia or a solid cancer, which solid cancer is preferably selected from the qroup of lymphoma, breast can ⁇ cer, prostate cancer, qastric cancer and ovarian cancer, more preferably epithelial ovarian cancer, even more preferably se ⁇ rous, mucinous or endometrioid epithelial ovarian cancer, espe ⁇ cially endometrioid epithelial ovarian cancer.
  • VAV3 was previ ⁇ ously shown as an oncoqene for leukemia, lymphoma, breast can ⁇ cer, prostate cancer, and qastric cancer but not ovarian cancer.
  • the cancer is a solid cancer
  • the cancer is endometrial cancer (see also Example 2) .
  • This endo ⁇ metrial cancer is preferably one of stage IA or IB, grade 1, stage IA or IB, grade 2, stage IA or IB, grade 3, any grade of stage II, any grade of stage IIIA, IIIB or IIIC or any grade of stage IVA or IVB, preferably stage IA or IB, grade 1, stage IA or IB, grade 2, stage IA or IB, grade 3, more preferably stage IA or IB grade 1 or stage IA or IB, grade 2, especially stage IA grade 1 (as it is beneficial to apply the present invention as early as possible, e.g.
  • the stages are taught e.g. in Pe- corelli.
  • the "grade” refers to a grade of a histopathological tumour grading scheme, for the purposes of the present invention a three-tier scheme of: low grade (1) - well-differentiated cells, intermediate grade (2), high grade (3) - poorly differen ⁇ tiated cells.
  • the cancer can also be lung cancer, liver cancer, stomach cancer, colorec ⁇ tal cancer, oesophageal cancer, bladder cancer, kidney cancer, melanoma, pancreatic cancer or thyroid cancer.
  • the cancer is epithelial ovarian cancer, it is preferably one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1 (as it is beneficial to apply the present invention as early as possible, e.g. in order to spare many patients an unnecessary chemother ⁇ apy) .
  • the stages are taught e.g. in Hennessy et al . 2009, panel 1.
  • the "grade” refers to a grade of a histopathological tumour grading scheme, for the purposes of the present invention a three-tier scheme of: low grade (1) - well-differentiated cells, intermediate grade (2), high grade (3) - poorly differentiated cells .
  • said cancer is a cancer prone to relapse, especially despite platinum-based, taxane-based or anti-angiogenic therapy, or com ⁇ binations thereof, preferably an ovarian cancer prone to re ⁇ lapse, especially despite platinum-based, taxane-based or anti- angiogenic therapy, or combinations thereof.
  • VAV3 is an excep ⁇ tionally suitable marker to predict relapse, especially ovarian cancer relapse and poor response to platinum-based taxane-based or anti-angiogenic therapy, or combinations thereof.
  • a method is pro ⁇ vided for diagnosing a cancer in a patient, comprising
  • the tissue sample can be a tumor tis ⁇ sue sample or a putative tumor tissue sample (see also Example 1 and Figs. 2-4);
  • the sample typically does not require special prepa ⁇ ration, it is preferably enriched for CSCs using methods relying on markers allowing their discrimination (more preferably en ⁇ richment is performed in a side population assay) .
  • the side population assay is disclosed e.g. in Boesch et al, 2014 and also in Golebiewska et al . , 2011.
  • Other markers allowing dis ⁇ crimination are disclosed e.g. in Medema 2013 (in particular Ta ⁇ ble 1) and comprise CD24, CD44, CD117 and CD133 for ovarian can ⁇ cer.
  • Methods to isolate cells displaying such markers are known in the art and comprise fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) .
  • FACS fluorescent-activated cell sorting
  • MCS magnetic-activated cell sorting
  • the sample is a body fluid, it is preferably enriched for tumor cells, in particular CSCs.
  • a preferred technique to detect VAV3 mRNA uses polynucleotide probes, especially labelled polynucleotide probes (e.g. fluorescent label, radioactive la ⁇ bel) that hybridize with the mRNA, preferably wherein the label is detected e.g. by a photometer, fluorometer or a scintillation counter.
  • a preferred technique to detect VAV3 protein uses antibodies, especially la ⁇ belled antibodies (e.g. fluorescent label, enzymatic label, ra ⁇ dioactive label) that bind to the protein, preferably wherein the label is detected e.g. by a photometer, fluorometer, a scin ⁇ tillation counter or a chemiluminescent-responsive or radiation- sensitive film.
  • VAV3 in particular VAV3var2
  • qRT- PCR quantitative real-time PCR
  • Other preferred techniques for detecting VAV3 (in particu ⁇ lar VAV3var2) mRNA or protein are immunohistochemical (IHC) methods, immunofluorescence (IF) methods, RNA in-situ hybridisa ⁇ tion. Further preferred methods comprise MALDI-MS (e.g.
  • WO 2009/004576 A including surface enhanced laser desorp- tion/ionization mass spectrometry (SELDI-MS) , especially sur ⁇ face-enhanced affinity capture (SEAC) , surface-enhanced need de- sorption (SEND) or surface-enhanced photo label attachment and release (SEPAR) ) , antibody testing (e.g.
  • WO 2008/031839 includ ⁇ ing immunoprecipitation, Western blotting, Enzyme-linked immuno sorbent assay (ELISA) , Enzyme-linked immuno sorbent assay (RIA) , dissociation-enhanced lanthanide fluoro immuno assay (DELFIA) , scintillation proximity assay (SPA) , and quantitative nucleic acid testing (e.g. in WO 2006/066965 A, especially PCR, LCR and RT-PCR) . It is also preferred to combine at least two of said techniques, e.g. for increased reliability.
  • nucleotide primers or antibodies are used in the techniques of the above paragraph. It is known in the art how to make primers or antibodies that bind VAV3var2 (see also Example for primers) .
  • sample of known can ⁇ cer or non-cancer status are usable in principle for comparison with the sample of unknown cancer status diagnosed according to the present invention.
  • Such reference, standard or control sam ⁇ ples can be taken e.g. from a patient with negative diagnosis regarding cancer.
  • a control sample, standard sample or reference sample is said to be comparable to the sample that is taken from a patient being suspected to be afflicted by ongoing cancer development according to the present invention, this means that both samples, except for their expression profile have been derived and treated equally.
  • the sample in both cases may e.g. be a tissue or body fluid sample which has been further treated in a way to allow for detection of the diagnos ⁇ tic marker molecules as mentioned above.
  • the mean amounts are calculated from more than one samples of known cancer or non-cancer state (especially preferred is when each of these samples is from a different subject) , preferably more than 3 samples, more preferably more than 5 samples, even more pref ⁇ erably more than 10 samples, especially more than 20 samples.
  • inventive method further com ⁇ prises diagnosing said patient as having said cancer if said measured amount is significantly higher than said mean amount of the negative control.
  • inventive method further comprises diagnosing said patient as having said cancer if said measured amount is essentially equal to or significantly higher to said mean amount of the positive control.
  • inventive method further comprises diagnosing said patient as not having said cancer if said measured amount is significantly lower than or essentially equal to said mean amount of the negative control.
  • inventive method further comprises diagnosing said patient as not having said cancer if said measured amount is significantly lower than said mean amount of the positive control.
  • the method is for predicting disease-free survival, event-free sur ⁇ vival, puncture-free survival and/or overall survival of the pa ⁇ tient .
  • Disease-free survival is defined as the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer, or time to death, which ever occurred first.
  • Event-free survival is defined as length of time after pri ⁇ mary treatment for a cancer ends that the patient remains free of certain complications or events that the treatment was in ⁇ tended to prevent or delay, or time to death, which ever oc ⁇ curred first. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from cancer that has spread to the bone.
  • Puncture-free survival is defined as time to first need for paracentesis after primary treatment, or time to death, which ever occurred first.
  • Another aspect of the present invention provides the use of an anti-VAV3var2 antibody or a functional fragment thereof for diagnosing a cancer in a patient or determining the state of a cancer patient with respect to cancer treatment.
  • the antibody of the present invention is used in the inventive method.
  • the antibody has an affinity to VAV3varl that is weaker than the antibody's affinity to VAV3var2, pref ⁇ erably at least 10 times weaker, more preferably at least 100 times weaker, especially at least 1000 times weaker. This will allow specific binding to the isoform that is the actual onco ⁇ genic factor.
  • the antibody has an affinity to VAV3varl that weaker, the same, or stronger than the antibody's affinity to VAV3var2, provided that it still binds both isoforms (VAV3varl and VAV3var2) .
  • the antibody binds to both isoforms and the dis ⁇ crimination between VAV3varl and VAV3var2 is based on their dif ⁇ ferent molecular weight (cf . the Western blot of Fig. 5) .
  • Functional fragments of antibodies are fragments that are also able to bind the respective epitope.
  • Such fragments are for instance Fab fragments, single-domain antibodies and binding do ⁇ mains derived from the constant region of an antibody (such as an FcabTM) .
  • the inventive fragments also comprise: "Kappa bodies” (III et al., Protein Eng. 10: 949-57 (1997)), “Minibodies” (Martin et al., EMBO J. 13: 5303-9 (1994)), “Diabodies” (Holliger et al . , Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or “Janusins” (Traunecker et al . , EMBO J. 10:3655-3659 (1991) and Traunecker et al . , Int. J. Cancer (Suppl.) 7:51-52 (1992)). They may be prepared using standard molecular biological techniques follow ⁇ ing the teachings of the specification.
  • a method to screen for candidate therapeutic compounds, especially for can ⁇ didate compounds for cancer therapy comprises :
  • the inventive method is a high-throughput screening method (cf . Maccarron 2011) . See for instance the fol ⁇ lowing documents for appropriate screening methods: Sundberg 2000, Mayr 2009, US 7815868 Bl, WO 2006/124318 A2.
  • the inventive method further com ⁇ prises designating as a candidate therapeutic compound, espe ⁇ cially as a candidate compound for cancer therapy, a compound of the at least one compounds, if said compound of the at least one compounds has a binding affinity of less than ⁇ , preferably less than ⁇ , more preferably less than ⁇ , especially less than InM to the VAV3var2 protein and/or if said compound of the at least one compounds significantly reduces VAV3var2 protein and/or VAV3var2 mRNA expression in the cell compared to a nega ⁇ tive control .
  • the inventive method is per ⁇ formed with the proviso that said designating is only performed if said compound of the at least one compounds has a binding af ⁇ finity to the VAV3varl protein that is higher in numeric value than the binding affinity to the VAV3var2 protein, preferably 10 times higher, more preferably 100 times higher, especially 1000 times higher and/or if said compound of the at least one com ⁇ pounds has essentially no effect on expression of VAV3varl pro- tein and/or VAV3varl mRNA expression in the cell. This ensures selectivity to the actual oncogenic factor, VAV3var2.
  • VAV3 is indicative of poor response to platinum-based chemother ⁇ apy, taxane-based chemotherapy or anti-angiogenic chemotherapy, or combinations thereof
  • a method of determining the state of a cancer patient with respect to cancer treatment comprises:
  • a platinum-based chemotherapeutic com ⁇ pound preferably cisplatin or carboplatin, or with an anti- angiogenic compound, preferably bevacizumab, or with a taxane compound, preferably paclitaxel, or combinations thereof.
  • the patient is not eligible for said treatment if said measured amount is significantly higher than said mean amount of the negative control.
  • the patient is not eligible for said treatment if said measured amount is essentially equal to or significantly higher to said mean amount of the positive control .
  • the patient is eligible for said treatment if said measured amount is significantly lower than or essentially equal to said mean amount of the negative control .
  • the patient is eligible for said treatment if said measured amount is significantly lower than said mean amount of the positive control.
  • VAV3var2 mRNA is quantitative real-time PCR (qRT-PCR) .
  • Other preferred techniques are immunohistochemical (IHC) methods, immunofluores- cence (IF) methods, RNA in-situ hybridisation.
  • Further preferred methods comprise MALDI-MS (e.g. WO 2009/004576 A (including sur ⁇ face enhanced laser desorption/ionization mass spectrometry
  • SEAC surface-enhanced affinity capture
  • SEND surface-enhanced need desorption
  • SEPAR surface-enhanced photo label attachment and release
  • SPA quantitative nucleic acid testing
  • WO 2006/066965 A especially PCR, LCR and RT-PCR
  • nucleotide primers or antibodies are used in the techniques of the above paragraph. It is known in the art how to make primers or antibodies that bind VAV3var2 (examples for such primers are mentioned herein) .
  • sample of known can ⁇ cer or non-cancer status are usable in principle for comparison with the sample of unknown cancer status diagnosed according to the present invention.
  • Such reference, standard or control sam ⁇ ples can be taken e.g. from a patient with negative diagnosis regarding cancer.
  • a control sample, standard sample or reference sample is said to be comparable to the sample that is taken from a patient being suspected to be afflicted by ongoing cancer development according to the present invention, this means that both samples, except for their expression profile have been derived and treated equally.
  • the sample in both cases may e.g. be a tissue or blood derived sample which has been further treated in a way to allow for detection of the di ⁇ agnostic marker molecules as mentioned above.
  • the mean amounts are calculated from more than one samples of known cancer or non-cancer state (especially preferred is when each of these samples is from a different subjects) , preferably more than 3 samples, more preferably more than 5 samples, even more preferably more than 10 samples, especially more than 20 sam ⁇ ples .
  • VAV3 is an isoform of VAV3, preferably VAV3var2.
  • said cancer is a solid can ⁇ cer, preferably selected from the group of lymphoma, breast can ⁇ cer, prostate cancer, gastric cancer, endometrial cancer and ovarian cancer, more preferably epithelial ovarian cancer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian can ⁇ cer .
  • said cancer is leukae ⁇ mia .
  • the term "significantly lower” means more than 10%, preferably more than 20%, more preferably more than 50% lower.
  • the term “significantly higher” means more than 10%, preferably more than 20%, more preferably more than 50%, even more preferably more than 100%, especially more than 200% higher .
  • the patient is pref ⁇ erably a mammal, more preferably a primate, especially a human.
  • the term "patient” in general, when not referring to a specific disease, e.g. "cancer patient"
  • the subject which is called patient is actually ill or at risk of an illness (e.g. cancer) .
  • the sub ⁇ ject is only suspected of having an illness (e.g. cancer) or, in fact, healthy.
  • the VAV3 protein (or isoforms or variants thereof) is of the same species as the patient.
  • the VAV3 protein (or isoforms or variants thereof) is of the same species as the patient.
  • the human VAV3varl mRNA sequence is preferably selected from one of SEQ ID NO: 1, NCBI RefSeq database entry NM_006113.4, and single- nucleotide polymorphisms thereof, in particular the human VAV3varl mRNA sequence is selected from SEQ ID NO: 1.
  • the human VAV3var2 mRNA sequence is preferably selected from one of SEQ ID NO: 2, NCBI RefSeq database entry NM_001079874.1, and single- nucleotide polymorphisms thereof, in particular the human VAV3var2 mRNA sequence is selected from SEQ ID NO: 2.
  • the human VAV3varl protein sequence is preferably selected from one of SEQ ID NO: 3, NCBI RefSeq database entry NP_006104.4, and single- amino-acid mutations thereof, in particular the human VAV3varl protein sequence is selected from SEQ ID NO: 3.
  • the human VAV3var2 protein sequence is preferably selected from one of SEQ ID NO: 4, NCBI RefSeq database entry NP_001073343.1, and single- amino-acid mutations thereof, in particular the human VAV3var2 portein sequence is selected from SEQ ID NO: 4.
  • the above- mentioned National Center for Biotechnology Information (NCBI) database entry identifiers include the entry version number ".N"; and relate to NCBI RefSeq database release 66, 7 July 2014.
  • predicting shall not be construed in an absolute sense, i.e. with the meaning, that with 100% certainty it can be predicted that a patient will definitely develop cancer or the cancer (of a patient having the cancer) has a certain outcome. Instead, the invention relates to asserting an increased risk of a patient for developing cancer or for having a certain outcome.
  • diagnosis a can ⁇ cer also shall not be construed in the sense that all cancer patients can be identified by the inventive method but instead in the sense that the patient may have a increased probability of having the cancer. Therefore, the inventive method can be a first lead to identify the cancer, possibly followed by more in ⁇ vasive tests.
  • Embodiment 1 Use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a can ⁇ cer marker;
  • chemotherapeutic compound is a platinum- based therapeutic compound, preferably cisplatin or carboplatin or an angiogenic inhibitor, preferably bevacizumab, or a taxane, preferably paclitaxel,
  • chemotherapeutic compound is a platinum- based therapeutic compound, preferably cisplatin or carboplatin; or
  • VAV3var2 protein or VAV3var2 mRNA for diagnosing of a cancer in a tissue sample or in a body fluid sample, wherein VAV3var2 protein or VAV3var2 mRNA is used.
  • Embodiment 2 The use of embodiment 1, wherein said cancer is a solid cancer, preferably selected from the group of lym ⁇ phoma, breast cancer, prostate cancer, gastric cancer and ovar ⁇ ian cancer, more preferably epithelial ovarian cancer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
  • Embodiment 3 The use of embodiment 2, wherein said cancer is an epithelial ovarian cancer and is one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1.
  • Embodiment 4 The use of embodiment 1, wherein said cancer is leukaemia.
  • Embodiment 5 The use of any one of embodiments 1-4, wherein said cancer is a cancer prone to relapse, especially de ⁇ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
  • an ovarian cancer prone to relapse especially de ⁇ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
  • Embodiment 6 A method for diagnosing a cancer in a pa- tient, comprising:
  • -said measured amount is essentially equal to or signifi ⁇ cantly higher to said mean amount of the positive control; or diagnosing said patient as not having said cancer if
  • Embodiment 7 The method of embodiment 6, wherein said can ⁇ cer is a leukemia or a solid cancer, which solid cancer is pref ⁇ erably selected from the group of lymphoma, breast cancer, pros ⁇ tate cancer, gastric cancer and ovarian cancer, more preferably epithelial ovarian cancer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endo ⁇ metrioid epithelial ovarian cancer.
  • Embodiment 8 The method of embodiment 7, wherein said can ⁇ cer is an epithelial ovarian cancer and is one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1.
  • Embodiment 9 The method of any one of embodiments 6-8, wherein said cancer is a cancer prone to relapse, especially de ⁇ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
  • an ovarian cancer prone to relapse especially de ⁇ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
  • Embodiment 10 The use of the method of any one of embodi ⁇ ments 6-9, for predicting disease-free survival, event-free sur ⁇ vival, puncture-free survival and/or overall survival of said patient .
  • Embodiment 11 Use of an anti-VAV3var2 antibody or a frag ⁇ ment thereof for diagnosing a cancer in a patient or determining the state of a cancer patient with respect to cancer treatment, preferably in the method of any one of embodiments 6-10 or 15- 18, wherein the VAV3var2 protein amount is measured, and/or preferably wherein the antibody has an affinity to VAV3varl that is weaker than the antibody's affinity to VAV3var2, preferably at least 10 times weaker, more preferably at least 100 times weaker, especially at least 1000 times weaker.
  • Embodiment 12 A method to screen for candidate therapeutic compounds, especially for candidate compounds for cancer ther ⁇ apy, the method comprising
  • Embodiment 13 The method of embodiment 12, further compris ⁇ ing
  • a compound of the at least one compounds if said compound of the at least one com ⁇ pounds has a binding affinity of less than ⁇ , preferably less than ⁇ , more preferably less than ⁇ , especially less than InM to the VAV3var2 protein and/or if said compound of the at least one compounds significantly reduces VAV3var2 protein and/or VAV3var2 mRNA expression in the cell compared to a nega ⁇ tive control .
  • Embodiment 14 The method of embodiment 13, with the proviso that said designating is only performed if said compound of the at least one compounds has a binding affinity to the VAV3varl protein that is higher in numeric value than the binding affin ⁇ ity to the VAV3var2 protein, preferably 10 times higher, more preferably 100 times higher, especially 1000 times higher and/or if said compound of the at least one compounds has essentially no effect on expression of VAV3varl protein and/or VAV3varl mRNA expression in the cell.
  • Embodiment 15 A method of determining the state of a cancer patient with respect to cancer treatment, comprising
  • a platinum-based chemotherapeutic compound preferably cisplatin or carboplatin
  • an anti-angiogenic compound preferably bevacizumab, or with a taxane, preferably paclitaxel, or combinations thereof, in particular designating the patient as being eligible for the treatment with a platinum-based chemo- therapeutic compound, preferably cisplatin or carboplatin; if -said measured amount is significantly higher than said mean amount of the negative control, and/or
  • Embodiment 16 The method of embodiment 15, wherein VAV3 is an isoform of VAV3, preferably VAV3var2.
  • Embodiment 17 The method of embodiments 15 or 16, wherein said cancer is a solid cancer, preferably selected from the group of lymphoma, breast cancer, prostate cancer, gastric can ⁇ cer and ovarian cancer, more preferably epithelial ovarian can ⁇ cer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
  • a solid cancer preferably selected from the group of lymphoma, breast cancer, prostate cancer, gastric can ⁇ cer and ovarian cancer, more preferably epithelial ovarian can ⁇ cer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
  • Embodiment 18 The method of embodiments 15 or 16, wherein said cancer is leukaemia.
  • FIG. 1 CSC-Specific VAV3var2 Expression in Ovarian Cancer.
  • Ovarian CSC were purified from the indicated cell lines using ABC drug transporter-dependent dye efflux (side population as ⁇ say) .
  • the stem cell properties of the isolated cells (such as clonogenicity, tumorigenicity and asymmetric division) were con ⁇ firmed in functional assays.
  • VAV3var2 expression in CSC and non- CSC (exhibiting neither dye efflux nor functional stem cell properties) was determined on transcript level using qRT-PCR analysis. Quantification was done by normalizing Ct to reference Ct (TBP) .
  • A2780 is a commercially available human ovarian carci ⁇ noma cell line.
  • A2780V is a variant cell line of A2780.
  • IGROV1 is a commercially available human ovarian epithelial cancer cell line.
  • TBP TATA-box-binding protein.
  • Ct cycle threshold.
  • FIG. 2 Selective upregulation of VAV3var2 in Ovarian Tu ⁇ mour Tissue.
  • TBP TATA-box-binding protein.
  • Ct cycle threshold.
  • FIG. 3 VAV3 and VAV3var2 Predict Poor Outcome in Ovarian Cancer Patients.
  • VAV3 (A), VAV3varl (B) and VAV3var2 (C) tran ⁇ script level was determined in tumor tissue from 147 ovarian cancer patients using qRT-PCR. The data were sub-divided into low- and high-expressing cases according to 50th percentile sta ⁇ tistics, and correlated to disease-free survival (left-hand pan- els) and overall survival (right-hand panels) . Statistical sig ⁇ nificance was determined using log-rank test.
  • VAV3var2 but not VAV3, predicts Disease-Free Sur ⁇ vival in Endometrioid Ovarian Cancer Cases.
  • VAV3 (left-hand panel) and VAV3var2 (right-hand panel) transcript level was de ⁇ termined in tumor tissue from 25 endometrioid ovarian cancer pa ⁇ tients using qRT-PCR.
  • the data were sub-divided into low- and high-expressing cases according to 50th percentile statistics, and correlated to disease-free survival.
  • Statistical signifi ⁇ cance was determined using log-rank test.
  • FIG. 5 This Western Blot demonstrates that, in contrast to leukocytes (Jurkat) where the longer VAV3 isoform, VAV3varl, is predominant, ovarian cancer cells (OVCAR-3) express truncated versions of VAV3 protein such as VAV3var2.
  • VAV3var2 levels are significantly higher in ovarian cancer patients refractory to standard platinum based therapy.
  • FIG. 7 VAV3var2 expression predicts poor outcome in endo ⁇ metrial cancer patients.
  • VAV3var2 transcript levels were deter ⁇ mined using qRT-PCR.
  • Quantification was done by normalizing Ct to reference Ct (TBP) .
  • TBP TATA-box-binding protein.
  • Ct cycle threshold. The data were sub-divided into low- and high-expressing cases according to 75 th percentile sta ⁇ tistics, and correlated to overall survival (right panel) .
  • Example 1 Example 1
  • VAV3 (comprising VAV3varl and VAV3var2) was selec ⁇ tively overexpressed in ovarian cancer stem cells (CSC) recov ⁇ ered from ovarian cancer cell lines. Subsequent qRT-PCR analysis confirmed this result, and further showed that of the two vari- ants only VAV3var2 was overexpressed in ovarian CSC (Figure 1) .
  • VAV3var2 ( Figure 3A) and VAV3var2 ( Figure 3C) , but not of VAV3varl ( Figure 3B) , statistically significantly correlated with poor disease-free survival (DFS) and poor overall survival (OS) .
  • sub-classification of the patient cohort into major ovarian cancer subtypes i.e., serous, mucinous, endo ⁇ metrioid
  • VAV3var2 show total VAV3, and in particular, VAV3var2 as predictors of platinum refractoriness and cancer re ⁇ currence in ovarian cancer.
  • Tumor tissue (Tu) and healthy control tissue (N) was obtained from patients undergoing debulking surgery. Tissue was flash-frozen and pulverized. RNA extraction, reverse transcription and qRT-PCR was performed as described below.
  • VAV3var2 and cancer outcome (Figs. 3 and 4) .
  • Tumor tissue was obtained from patients undergoing debulking surgery. Tissue was flash-frozen and pulverized. RNA extraction, reverse transcrip ⁇ tion and qRT-PCR was performed as described below. 50 th percen ⁇ tile statistics was used to divide the cohort into low- and high-expressing cases, and DFS and OS were analysed using the Kaplan-Meier method. A total of 147 ovarian cancer patients were included in this study with a median age of 63 at diagnosis. 72% were in advanced FIGO stages 3/4; roughly 50% had serous, 30% mucinous and 17% endometrioid subtypes.
  • VAV3 isoform expression in lymphocytes and ovarian cancer cells (Fig. 5) : Protein fractions were harvested from ovarian cancer cells using a lysis buffer containing NP-40 as detergent and protease inhibitors for prevention of protein degradation. 25 pg of denatured protein were loaded per slot in reducing sample buffer (purchased from Life Technologies) and separated for 90 min at 150 V on a prefabricated SDS-PAGE gel (NuPAGE; purchased from Life Technolgies) . MOPS SDS was used as running buffer (purchased from Life Technologies) . Separated protein fractions were blotted onto a standard nitrocellulose membrane (120 min at 30 V) .
  • the nitrocellulose membrane was washed in TTBS and incu ⁇ bated for 60 min in 1% (w/v) milk powder to block unspecific binding sites. After washing, blotted proteins were incubated overnight with a rabbit anti-human VAV3 antibody (clone EP1130Y; purchased from Novus Biologicals) . The next day, the membrane was washed in TTBS and incubated for 60 min with a HRP-linked polyclonal anti-rabbit antibody (purchased from Cell Signaling) . Proteins were finally detected by enhanced chemiluminescence us ⁇ ing commercially available reagents (LumiGlo/Peroxid Reagent; purchased from Cell Signaling) . Alpha-tubulin was used as load ⁇ ing control .
  • A2780 cells were purchased from Sigma- Aldrich, Vienna, Austria.
  • OVCAR-3 cells were purchased from ATCC, Wesel, Germany.
  • IGROV1 cells were kindly obtained from Prof. R. Brown, London, UK, and the A2780 variant cell line A2780V was generously provided by Prof. R. Zeillinger, Vienna, Cells were maintained in appropriate culture medium at 37° in a humidified atmosphere containing 5% C02. Cells were harvested using trypsin and prepared for qRT-PCR as described above.
  • RNA was spun down for 8 min at 12, 000 g at 4°C and the supernatant was removed.
  • RNA concentration was determined using NanoDropTM Lite.
  • RNA reverse transcriptase was reverse transcriptase reverse transcriptase and random hexamer priming.
  • M-MLV Moloney Murine Leukaemia Virus
  • Mix I was incubated for 5 min at 65°C and immediately chilled on ice.
  • Mix II was added to Mix I, yielding a final reaction volume of 40 ⁇ .
  • the reaction was performed as follows: 10 min at 25°C (primer annealing), 60 min at 37°C (reverse transcrip ⁇ tion), 15 min at 70°C (heat inactivation) , and hold at 4°C.
  • qRT-PCR of cDNA templates was done using the ABI PRISM ® 7900HT Sequence Detection System and corresponding software (SDS 2.3). Amplification products were detected using fluorescently- labeled oligonucleotide hybridization probes (FAM/BHQ, FAM/TAMRA) . Relative quantification of gene expression was done by normalizing the respective C t values to reference C t value (TBP) .
  • the reaction mix contained the following components:
  • Primer F (10 pM) 2,25 900 nM
  • Primer R (10 pM) 2,25 900 nM
  • the reaction was performed as follows: 2 min at 50°C, 10 min at 95° C (denaturing) , and 45 amplification cycles comprising 15 sec at 95°C (denaturing) and 1 min at 60°C (primer annealing and
  • VAV3 Sense VAV3ALL_s(1)_A 8 CCA GAG AAA CGG ACC AAT GG
  • Antisense VAV3var1+2_as(1)_A 13 TCC ACC TGT TTA GGA GTT CTT CG
  • Tumor tissue and, where possible, healthy adjoining tissue was obtained from endometrial cancer patients undergoing debulking surgery. Tissue was flash-frozen and pulverized. RNA extraction, reverse transcription and qRT-PCR was performed as described in Example 1. 75 th percentile statistics was used to divide the co ⁇ hort into low- and high-expressing cases, and overall survival was analysed using the Kaplan-Meier method. A total of 90 endo ⁇ metrial cancer patients were included in this study. VAV3var2 expression predicts poor outcome in endometrial cancer patients, as shown in Fig. 7.

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Abstract

The present invention provides the use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a cancer marker, especially for ovarian cancer or endometrial cancer, as well as a use of an anti-VAV3var2 antibody. Further, a method relating to VAV3, for treatment decisions in regard to platinum-based chemotherapy, taxane-based chemotherapy and anti-angiogenic chemotherapy is provided as well as a method to screen for candidate therapeutic compounds, relating to VAV3var2.

Description

VAV3 as a marker for cancer
The present invention relates to the field of biomarkers for diagnosing cancer, especially ovarian cancer or endometrial can¬ cer .
Cancers are a leading cause of death worldwide, causing more than 8 million deaths in 2012 (WHO fact sheet No. 297 on cancer, as of February 2014) . Cancer is a generic term for a large group of diseases that can affect any part of the body. Other terms used are malignant tumours and neoplasms. One defining feature of cancer is the rapid creation of abnormal cells that grow be¬ yond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. This process is referred to as metastasis. Metastases are the major cause of death from cancer.
Ovarian cancer is a frequent form of cancer, 239000 inci¬ dences and 152000 deaths of women were estimated for the year 2012. By far the most abundant form of ovarian cancer is epithe¬ lial ovarian cancer which results from malignant transformation of the ovarian surface epithelium (Hennessy et al . , 2009) . The lifetime incidence risk is about 1 in 70 (women) . Identifiable genetic predispositions such as BRCA1/BRCA2 mutations are pre¬ sent in only 10-15% of patients. Epithelial ovarian cancers are typically classified by histopathological grade (1-3), stages (I-IV) and appearance into serous (most common), mucinous and endometrioid subtypes. Hennessy et al . give an overview over the standard of care for epithelial ovarian cancer. If epithelial ovarian cancer is suspected on the basis of physical examination and imaging, an exploratory laparotomy is usually performed for histological confirmation and tumor debulking, i.e. the partial removal of tumor tissue (also known as cytoreduction) . For many patients with early stage disease with well-differentiated tu¬ mors (i.e. grade 1), systemic therapy with chemotherapeutic com¬ pounds (which has severe side-effects) is not warranted, as 90% of them have long-term disease free survival without chemothera- peutic treatment. Therefore, and because 50-60% of patients with high-risk early stage disease will not have recurrence if moni¬ tored, there is a crucial need to identify molecular markers that will allow accurate identification of patients who will benefit from chemotherapy. Carboplatin (or platinum-based therapies in general) (Arm¬ strong et al . , 2006), in combination with paclitaxel (a taxane) are an effective treatment for ovarian cancer. In addition the angiogenesis inhibitor bevacizumab has been introduced recently (Perren et al . , 2011) . Yet some patients have ovarian cancers that only respond insufficiently (e.g. refractory cancers) to treatment with platinum-based chemotherapeutic compounds and bevacizumab (or angiogenic inhibitors in general) . Again, there is a crucial need for molecular markers that can predict treat¬ ment success of such compounds and thereby inform the physician on what treatment options to choose (e.g. whether to use plati¬ num-based chemotherapeutic compounds and/or angiogenic inhibi¬ tors for systemic therapy) .
Taken together, mortality in ovarian cancer (and certain other cancers) remains high because no or only few sufficiently robust molecular markers (also called biomarkers herein) are known. In addition, biomarkers that give a clear guidance for what treatment options to choose (i.e. targeted therapy) are even harder to come by.
Early diagnosis and targeted therapy are key for successful treatment of cancer (especially in terms of longer patient sur¬ vival) .
Hence, it is an object of the present invention to provide a biomarker for diagnosing cancer, in particular ovarian cancer, as well as for guidance for targeted treatment, especially in regard to treatment with angiogenic inhibitors, platinum-based chemotherapeutic compounds or taxanes, or combinations thereof.
Therefore, the present invention provides the use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a cancer marker.
VAV3 is a guanine nucleotide exchange factor for Rho/Rac GTPases having core functions in cytoskeletal rearrangements and cell migration (Hornstein et al . , 2004) . VAV3 has oncogenic properties in a variety of tumor entities, including leukemia, lymphoma, breast cancer, prostate cancer, and gastric cancer (cf . e.g. Chang et al . , 2012 and Lin et al . , 2012 A) . VAV3 ex¬ pression increases during prostate cancer progression, and high expression of VAV3 also correlates with prostate cancer recur- rence (Lin et al . , 2012 B) .
Of note, VAV3 comprises the transcript variants VAV3varl (NCBI RefSeq entry NM_006113.4 for H. sapiens) and VAV3var2 (NCBI RefSeq entry NM_001079874.1 for H. sapiens), whose expres¬ sion has never been separately investigated in a cancer setting.
Cancer stem cells (CSC) are believed to be involved in tumor evasion subsequent to or during classical antitumor therapies and have thus become an attractive target for further improve¬ ment of anticancer strategies (Boesch et al . , 2014) . A tran¬ script or protein that is overexpressed in CSCs may be a suit¬ able cancer marker. The relevance of CSCs in cancer is also dis¬ cussed in Pattabiraman & Weinberg, 2014.
In the course of the present invention, it was surprisingly found that total VAV3 (comprising VAV3varl and VAV3var2) was se¬ lectively overexpressed in ovarian cancer stem cells recovered from ovarian cancer cell lines. However, even more surprisingly, only VAV3var2 was overexpressed in ovarian CSCs (Fig. 1) . Thus, it was shown for the first time that tumoral VAV3 expression is almost exclusively due to expression of VAV3var2. Furthermore, it was found that VAV3 and especially VAV3var2 are a robust pre¬ dictor of patient outcome in ovarian cancer, in particular in regard to relapse in general, and/or response to platinum-based chemotherapies, taxane-based chemotherapies and/or anti- angiogenic chemotherapies.
This is also due to the fact that CSCs are multidrug- resistant. VAV3 (especially VAV3var2) being a robust marker for CSCs - as found in the course of the present invention - is hence also a marker for drug-resistance such as insufficient re¬ sponse to the chemotherapies mentioned in the paragraph above. This applies especially to anti-angiogenetic inhibitors, as VAV3 has an important function in tumor angiogenesis (cf. e.g. Hunter et al, 2006 and Brantley-Sieders et al . , 2009).
VAV3 (but not VAV3var2) is known from the following cancer- related patent applications:
The WO 2012/066451 Al relates to prognostic and predictive gene signatures for colon cancer.
The WO 2013/052480 Al relates to a marker-based prognostic risk score in colon cancer. The WO 2010/145796 A2 relates to biomarkers and methods for determining efficacy of anti-EGFR antibodies in cancer therapy.
The WO 2007/025044 A2 relates to biomarkers and methods for determining sensitivity to epidermal growth factor receptor modulators .
The WO 2013/049398 A2 recites VAV3 as one of many biomarkers for cancer.
Neither of the above-identified patent applications disclose a specific relation of VAV3 to ovarian cancer, or VAV3 variants or isoforms. All of said applications are silent on treatment decisions relating to platinum-based chemotherapeutics or angio- genesis inhibitors.
The EP 2 388 336 Al relates to methods and kits for diagnos¬ ing colorectal cancer. Paragraph [0045] of the document refers to Table 1 of the document for 2002 RNAs that are indicative of the presence or absence of colorectal cancer. The entry refer¬ ring to SEQ ID NO: 108 in the table contains the identifiers "220131_at", "FXYD7 " and "NM_022006.1", all of which are in fact related to the FXYD domain-containing ion transport regulator 7 (FXYD7) . However, SEQ ID NO: 108 of the sequence listing of EP 2 388 336 Al contains the VAV3var2 transcript sequence, which is clearly a mistake as FXYD7 is another gene. Consequently, VAV3var2 is not disclosed or suggested as a cancer marker in the document .
Trenkle et al . relates to major transcript variants of VAV3, one of them being termed "VAV3.1". The length of human VAV3.1 is disclosed to be 3062 bases (p. 145, left column, 2nd paragraph of the document) , which is shorter than the VAV3var2 transcript (3115 bases, cf . SEQ ID NO: 2) . In addition, the divergence point disclosed for VAV3.1 (Fig. 1 of the document) is different from the one of the VAV3var2 transcript. Furthermore, the docu¬ ment does not disclose VAV3.1 as a cancer marker for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient or for diagnosing of a cancer in a tissue sample or in a body fluid sample. In ad¬ dition, VAV3.1 is disclosed to be expressed in normal colon and only a few tumor cell lines (Fig. 5 of the document), it is not even disclosed to be expressed in both ovarian cancer cell lines tested. Notwithstanding that VAV3.1 as disclosed in the document is not VAV3var2, the skilled person would hence not draw the conclusion from the document that VAV3.1 is a cancer marker. In conclusion, the document does not anticipate or suggest the pre¬ sent invention.
Qi et al. speculate that VAV3var2 (NM_001079874, called VAV3.1 in Qi et al . ) is an oncogene. However, the document does not disclose VAV3var2 as a cancer marker for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient or for diagnosing of a cancer in a tissue sample or in a body fluid sample. In addition, Qi et al . does not relate to ovarian cancer. Specifically, in Fig. 5C of the document a reduction of transcription of VAV3var2 upon addition of astragaloside IV, a compound asserted to be antipro¬ liferative, to HepG2 cells is shown. However, the skilled person would not draw from this the conclusion that VAV3var2 is a can¬ cer marker for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient or for diagnosing of a cancer in a tissue sample or in a body fluid sample.
For at least the above reasons, the present invention is the first to provide the use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a can¬ cer marker, said use being for determining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient, preferably wherein said chemotherapeutic compound is a platinum-based therapeutic compound, preferably cisplatin or carboplatin, or an angiogenic inhibitor, preferably bevacizumab, or a taxane, or combinations thereof.
A cancer sample can also be obtained by biopsy of tumours of said patient or, in the case of liquid tumours, by obtaining body fluids from said patient. For ovarian cancer, such a biopsy is typically a exploratory laparotomy of the tumour tissue (Hennessy et al . , 2009) . Preferably, the patient has been diag¬ nosed or is suspected of having cancer before applying the in¬ ventive use of treatment determination.
Platinum based compounds are widely used in chemotherapy. They comprise cisplatin, carboplatin, oxaliplatin (US 4,169,846), satraplatin (e.g. WO 2005/077385 A2), picoplatin, nedaplatin and triplatin.
Angiogenic inhibitors (or anti-angiogenic compounds) are also widely used in chemotherapy. The most prominent one is bevacizumab which is an inhibitor of the VEGF pathway (see e.g. WO 1994/010202 Al, WO 1998/045331 A2 ) .
Taxanes are widely used in chemotherapy as well. The most prominent taxanes are paclitaxel and docetaxel.
Said inventive use is alternatively for diagnosing of a can¬ cer in a tissue sample or in a body fluid sample, wherein VAV3var2 protein or VAV3var2 mRNA is used. It is evident that the tissue sample can be a tumor tissue sample or a putative tu¬ mor tissue sample (see also Example 1 and Figs. 2-4) .
VAV3varl is significantly longer than VAV3var2 on transcript level (4,776 bp versus 3,115 bp) . This size difference is due to significant truncation of VAV3var2 at the 5' -end, whereas 96% of the remaining sequence is identical to VAV3varl . On protein level, VAV3varl (NCBI Protein database entry NP_006104.4 for H. sapiens) is also siqnificantly lonqer than VAV3var2 (NCBI Pro¬ tein database entry NP_001073343.1 for H. sapiens) (847 aa ver¬ sus 287 aa) .
VAV3 belonqs to the evolutionary conserved VAV family of qua- nine nucleotide exchanqe factors, which also includes the VAV proteins VAV1 and VAV2. Thus, VAV1-VAV3 share sequence homoloqy which may also result in partially overlappinq protein function. However, in the qene array analysis made in the course of the present invention, VAV1 and VAV2 (in contrast to VAV3) did not show differential expression between CSC and non-CSC.
Preferably, in the entire context of the present invention, the cancer is a leukemia or a solid cancer, which solid cancer is preferably selected from the qroup of lymphoma, breast can¬ cer, prostate cancer, qastric cancer and ovarian cancer, more preferably epithelial ovarian cancer, even more preferably se¬ rous, mucinous or endometrioid epithelial ovarian cancer, espe¬ cially endometrioid epithelial ovarian cancer. VAV3 was previ¬ ously shown as an oncoqene for leukemia, lymphoma, breast can¬ cer, prostate cancer, and qastric cancer but not ovarian cancer.
In a further preference, if the cancer is a solid cancer, for embodiments in the entire context of the present invention the cancer is endometrial cancer (see also Example 2) . This endo¬ metrial cancer is preferably one of stage IA or IB, grade 1, stage IA or IB, grade 2, stage IA or IB, grade 3, any grade of stage II, any grade of stage IIIA, IIIB or IIIC or any grade of stage IVA or IVB, preferably stage IA or IB, grade 1, stage IA or IB, grade 2, stage IA or IB, grade 3, more preferably stage IA or IB grade 1 or stage IA or IB, grade 2, especially stage IA grade 1 (as it is beneficial to apply the present invention as early as possible, e.g. in order to spare many patients an un¬ necessary chemotherapy) . The stages are taught e.g. in Pe- corelli. The "grade" refers to a grade of a histopathological tumour grading scheme, for the purposes of the present invention a three-tier scheme of: low grade (1) - well-differentiated cells, intermediate grade (2), high grade (3) - poorly differen¬ tiated cells.
In the entire context of the present invention, the cancer can also be lung cancer, liver cancer, stomach cancer, colorec¬ tal cancer, oesophageal cancer, bladder cancer, kidney cancer, melanoma, pancreatic cancer or thyroid cancer.
In the entire context of the present invention, if the cancer is epithelial ovarian cancer, it is preferably one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1 (as it is beneficial to apply the present invention as early as possible, e.g. in order to spare many patients an unnecessary chemother¬ apy) . The stages are taught e.g. in Hennessy et al . 2009, panel 1. The "grade" refers to a grade of a histopathological tumour grading scheme, for the purposes of the present invention a three-tier scheme of: low grade (1) - well-differentiated cells, intermediate grade (2), high grade (3) - poorly differentiated cells .
Beneficially, in the entire context of the present invention, said cancer is a cancer prone to relapse, especially despite platinum-based, taxane-based or anti-angiogenic therapy, or com¬ binations thereof, preferably an ovarian cancer prone to re¬ lapse, especially despite platinum-based, taxane-based or anti- angiogenic therapy, or combinations thereof. It has been shown in the course of the present invention that VAV3 is an excep¬ tionally suitable marker to predict relapse, especially ovarian cancer relapse and poor response to platinum-based taxane-based or anti-angiogenic therapy, or combinations thereof.
In another aspect of the present invention, a method is pro¬ vided for diagnosing a cancer in a patient, comprising
-providing a tissue sample or a body fluid sample of said pa¬ tient, it is evident that the tissue sample can be a tumor tis¬ sue sample or a putative tumor tissue sample (see also Example 1 and Figs. 2-4); and
-measuring the amount of VAV3var2 protein or the amount of VAV3var2 mRNA in said sample; and
-comparing said amount with a mean amount of VAV3var2 pro¬ tein, or a mean amount of VAV3var2 mRNA, present in one or more samples of known cancer state as a positive control, and/or with a mean amount of VAV3var2 protein, or a mean amount of VAV3var2 mRNA, present in one or more samples of known non-cancer state as a negative control.
Although the sample typically does not require special prepa¬ ration, it is preferably enriched for CSCs using methods relying on markers allowing their discrimination (more preferably en¬ richment is performed in a side population assay) . The side population assay is disclosed e.g. in Boesch et al, 2014 and also in Golebiewska et al . , 2011. Other markers allowing dis¬ crimination are disclosed e.g. in Medema 2013 (in particular Ta¬ ble 1) and comprise CD24, CD44, CD117 and CD133 for ovarian can¬ cer. Methods to isolate cells displaying such markers are known in the art and comprise fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) .
Especially when the sample is a body fluid, it is preferably enriched for tumor cells, in particular CSCs.
A preferred technique to detect VAV3 mRNA (in particular VAV3var2 mRNA) uses polynucleotide probes, especially labelled polynucleotide probes (e.g. fluorescent label, radioactive la¬ bel) that hybridize with the mRNA, preferably wherein the label is detected e.g. by a photometer, fluorometer or a scintillation counter. A preferred technique to detect VAV3 protein (in par- ticular VAV3var2 protein) uses antibodies, especially la¬ belled antibodies (e.g. fluorescent label, enzymatic label, ra¬ dioactive label) that bind to the protein, preferably wherein the label is detected e.g. by a photometer, fluorometer, a scin¬ tillation counter or a chemiluminescent-responsive or radiation- sensitive film.
Particularly preferred for measuring the amount of VAV3 (in particular VAV3var2) mRNA is quantitative real-time PCR (qRT- PCR) . Other preferred techniques for detecting VAV3 (in particu¬ lar VAV3var2) mRNA or protein are immunohistochemical (IHC) methods, immunofluorescence (IF) methods, RNA in-situ hybridisa¬ tion. Further preferred methods comprise MALDI-MS (e.g. WO 2009/004576 A (including surface enhanced laser desorp- tion/ionization mass spectrometry (SELDI-MS) , especially sur¬ face-enhanced affinity capture (SEAC) , surface-enhanced need de- sorption (SEND) or surface-enhanced photo label attachment and release (SEPAR) ) , antibody testing (e.g. WO 2008/031839 (includ¬ ing immunoprecipitation, Western blotting, Enzyme-linked immuno sorbent assay (ELISA) , Enzyme-linked immuno sorbent assay (RIA) , dissociation-enhanced lanthanide fluoro immuno assay (DELFIA) , scintillation proximity assay (SPA) , and quantitative nucleic acid testing (e.g. in WO 2006/066965 A, especially PCR, LCR and RT-PCR) . It is also preferred to combine at least two of said techniques, e.g. for increased reliability.
Preferably, nucleotide primers or antibodies are used in the techniques of the above paragraph. It is known in the art how to make primers or antibodies that bind VAV3var2 (see also Example for primers) .
All reference, standard or control (these words are synonyms for the purposes of the present invention) samples of known can¬ cer or non-cancer status are usable in principle for comparison with the sample of unknown cancer status diagnosed according to the present invention. Such reference, standard or control sam¬ ples can be taken e.g. from a patient with negative diagnosis regarding cancer. If such a control sample, standard sample or reference sample is said to be comparable to the sample that is taken from a patient being suspected to be afflicted by ongoing cancer development according to the present invention, this means that both samples, except for their expression profile have been derived and treated equally. Thus, the sample in both cases may e.g. be a tissue or body fluid sample which has been further treated in a way to allow for detection of the diagnos¬ tic marker molecules as mentioned above.
The mean amount can also be calculated from a single sample (i.e. N=l), for instance it is possible to use healthy adjoining tissue of the patient as negative control. Beneficially, the mean amounts are calculated from more than one samples of known cancer or non-cancer state (especially preferred is when each of these samples is from a different subject) , preferably more than 3 samples, more preferably more than 5 samples, even more pref¬ erably more than 10 samples, especially more than 20 samples.
In a preferred embodiment the inventive method further com¬ prises diagnosing said patient as having said cancer if said measured amount is significantly higher than said mean amount of the negative control.
In another preferred embodiment the inventive method further comprises diagnosing said patient as having said cancer if said measured amount is essentially equal to or significantly higher to said mean amount of the positive control.
In another preferred embodiment the inventive method further comprises diagnosing said patient as not having said cancer if said measured amount is significantly lower than or essentially equal to said mean amount of the negative control.
In another preferred embodiment the inventive method further comprises diagnosing said patient as not having said cancer if said measured amount is significantly lower than said mean amount of the positive control.
In another preferred embodiment of the present invention, the method is for predicting disease-free survival, event-free sur¬ vival, puncture-free survival and/or overall survival of the pa¬ tient .
Disease-free survival is defined as the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer, or time to death, which ever occurred first.
Event-free survival is defined as length of time after pri¬ mary treatment for a cancer ends that the patient remains free of certain complications or events that the treatment was in¬ tended to prevent or delay, or time to death, which ever oc¬ curred first. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from cancer that has spread to the bone.
Puncture-free survival is defined as time to first need for paracentesis after primary treatment, or time to death, which ever occurred first.
Overall survival is defined as time to death after primary treatment has ended.
Another aspect of the present invention provides the use of an anti-VAV3var2 antibody or a functional fragment thereof for diagnosing a cancer in a patient or determining the state of a cancer patient with respect to cancer treatment. Preferably, the antibody of the present invention is used in the inventive method. Beneficially, the antibody has an affinity to VAV3varl that is weaker than the antibody's affinity to VAV3var2, pref¬ erably at least 10 times weaker, more preferably at least 100 times weaker, especially at least 1000 times weaker. This will allow specific binding to the isoform that is the actual onco¬ genic factor.
In another preferred embodiment of the present invention, the antibody has an affinity to VAV3varl that weaker, the same, or stronger than the antibody's affinity to VAV3var2, provided that it still binds both isoforms (VAV3varl and VAV3var2) . In this embodiment, the antibody binds to both isoforms and the dis¬ crimination between VAV3varl and VAV3var2 is based on their dif¬ ferent molecular weight (cf . the Western blot of Fig. 5) .
Functional fragments of antibodies are fragments that are also able to bind the respective epitope. Such fragments are for instance Fab fragments, single-domain antibodies and binding do¬ mains derived from the constant region of an antibody (such as an Fcab™) .
The inventive fragments also comprise: "Kappa bodies" (III et al., Protein Eng. 10: 949-57 (1997)), "Minibodies" (Martin et al., EMBO J. 13: 5303-9 (1994)), "Diabodies" (Holliger et al . , Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)), or "Janusins" (Traunecker et al . , EMBO J. 10:3655-3659 (1991) and Traunecker et al . , Int. J. Cancer (Suppl.) 7:51-52 (1992)). They may be prepared using standard molecular biological techniques follow¬ ing the teachings of the specification.
An overview over such engineered antibody fragments that are suitable in the present invention is also given in Hollinger & Hudson (Nat Biotechnol. 2005, PMID: 16151406).
In another aspect of the present invention, a method to screen for candidate therapeutic compounds, especially for can¬ didate compounds for cancer therapy is provided. This method comprises :
-providing at least one compound and
-measuring the binding affinity of the at least one compound to VAV3var2 protein or measuring the effect of the at least one compound on VAV3var2 protein or mRNA expression in a cell.
Techniques for screening of chemical compound libraries is known in the art, for both solution-based and cell-based ap¬ proaches. Preferably, the inventive method is a high-throughput screening method (cf . Maccarron 2011) . See for instance the fol¬ lowing documents for appropriate screening methods: Sundberg 2000, Mayr 2009, US 7815868 Bl, WO 2006/124318 A2.
In a preferred embodiment, the inventive method further com¬ prises designating as a candidate therapeutic compound, espe¬ cially as a candidate compound for cancer therapy, a compound of the at least one compounds, if said compound of the at least one compounds has a binding affinity of less than ΙΟΟΟηΜ, preferably less than ΙΟΟηΜ, more preferably less than ΙΟηΜ, especially less than InM to the VAV3var2 protein and/or if said compound of the at least one compounds significantly reduces VAV3var2 protein and/or VAV3var2 mRNA expression in the cell compared to a nega¬ tive control .
In another preferred embodiment, the inventive method is per¬ formed with the proviso that said designating is only performed if said compound of the at least one compounds has a binding af¬ finity to the VAV3varl protein that is higher in numeric value than the binding affinity to the VAV3var2 protein, preferably 10 times higher, more preferably 100 times higher, especially 1000 times higher and/or if said compound of the at least one com¬ pounds has essentially no effect on expression of VAV3varl pro- tein and/or VAV3varl mRNA expression in the cell. This ensures selectivity to the actual oncogenic factor, VAV3var2.
As it was found in the course of the present invention that VAV3 is indicative of poor response to platinum-based chemother¬ apy, taxane-based chemotherapy or anti-angiogenic chemotherapy, or combinations thereof, in another aspect of the present inven¬ tion a method of determining the state of a cancer patient with respect to cancer treatment is provided. This method comprises:
-measuring the amount of VAV3 mRNA or protein expression in a cancer biopsy sample of the patient; and
-comparing said amount with a mean amount of VAV3 protein, or a mean amount of VAV3 mRNA, present in one or more samples of known cancer state as a positive control, and/or with a mean amount of VAV3 protein, or a mean amount of VAV3 mRNA, present in one or more samples of known non-cancer state as a negative control; and
-designating the patient as being eligible or not eligible for the treatment with a platinum-based chemotherapeutic com¬ pound, preferably cisplatin or carboplatin, or with an anti- angiogenic compound, preferably bevacizumab, or with a taxane compound, preferably paclitaxel, or combinations thereof.
In a preferred embodiment the patient is not eligible for said treatment if said measured amount is significantly higher than said mean amount of the negative control.
In another preferred embodiment the patient is not eligible for said treatment if said measured amount is essentially equal to or significantly higher to said mean amount of the positive control .
In another preferred embodiment the patient is eligible for said treatment if said measured amount is significantly lower than or essentially equal to said mean amount of the negative control .
In another preferred embodiment the patient is eligible for said treatment if said measured amount is significantly lower than said mean amount of the positive control.
Particularly preferred for measuring the amount of VAV3var2 mRNA is quantitative real-time PCR (qRT-PCR) . Other preferred techniques are immunohistochemical (IHC) methods, immunofluores- cence (IF) methods, RNA in-situ hybridisation. Further preferred methods comprise MALDI-MS (e.g. WO 2009/004576 A (including sur¬ face enhanced laser desorption/ionization mass spectrometry
(SELDI-MS) , especially surface-enhanced affinity capture (SEAC) , surface-enhanced need desorption (SEND) or surface-enhanced photo label attachment and release (SEPAR) ) , antibody testing
(e.g. WO 2008/031839 (including immunoprecipitation, Western blotting, Enzyme-linked immuno sorbent assay (ELISA) , Enzyme- linked immuno sorbent assay (RIA) , dissociation-enhanced lantha- nide fluoro immuno assay (DELFIA) , scintillation proximity assay
(SPA), and quantitative nucleic acid testing (e.g. in WO 2006/066965 A, especially PCR, LCR and RT-PCR) . It is also pre¬ ferred to combine at least two of said techniques, e.g. for in¬ creased reliability.
Preferably, nucleotide primers or antibodies are used in the techniques of the above paragraph. It is known in the art how to make primers or antibodies that bind VAV3var2 (examples for such primers are mentioned herein) .
All reference, standard or control (these words are synonyms for the purposes of the present invention) samples of known can¬ cer or non-cancer status are usable in principle for comparison with the sample of unknown cancer status diagnosed according to the present invention. Such reference, standard or control sam¬ ples can be taken e.g. from a patient with negative diagnosis regarding cancer. If such a control sample, standard sample or reference sample is said to be comparable to the sample that is taken from a patient being suspected to be afflicted by ongoing cancer development according to the present invention, this means that both samples, except for their expression profile have been derived and treated equally. Thus, the sample in both cases may e.g. be a tissue or blood derived sample which has been further treated in a way to allow for detection of the di¬ agnostic marker molecules as mentioned above.
The mean amount can also be calculated from a single sample (i.e. N=l), for instance it is possible to use healthy adjoining tissue of the patient as negative control. Beneficially, the mean amounts are calculated from more than one samples of known cancer or non-cancer state (especially preferred is when each of these samples is from a different subjects) , preferably more than 3 samples, more preferably more than 5 samples, even more preferably more than 10 samples, especially more than 20 sam¬ ples .
In a particularly preferred embodiment, VAV3 is an isoform of VAV3, preferably VAV3var2.
In another preferred embodiment, said cancer is a solid can¬ cer, preferably selected from the group of lymphoma, breast can¬ cer, prostate cancer, gastric cancer, endometrial cancer and ovarian cancer, more preferably epithelial ovarian cancer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian can¬ cer .
In yet another preferred embodiment, said cancer is leukae¬ mia .
As used herein, the expression "A essentially equal to B" re¬ fers to A having the same value as B or A being within a -10% to +10% range of B (wherein the percentage is calculated from B) .
As used herein, the term "significantly lower" means more than 10%, preferably more than 20%, more preferably more than 50% lower. The term "significantly higher" means more than 10%, preferably more than 20%, more preferably more than 50%, even more preferably more than 100%, especially more than 200% higher .
In the context of the present invention, the patient is pref¬ erably a mammal, more preferably a primate, especially a human. Herein, the term "patient" (in general, when not referring to a specific disease, e.g. "cancer patient"), is not to be construed that the subject which is called patient is actually ill or at risk of an illness (e.g. cancer) . It can also mean that the sub¬ ject is only suspected of having an illness (e.g. cancer) or, in fact, healthy.
Preferably, as used herein, the VAV3 protein (or isoforms or variants thereof) is of the same species as the patient.
Preferably, as used herein, the VAV3 protein (or isoforms or variants thereof) is of the same species as the patient.
In the entire context of the present invention: The human VAV3varl mRNA sequence is preferably selected from one of SEQ ID NO: 1, NCBI RefSeq database entry NM_006113.4, and single- nucleotide polymorphisms thereof, in particular the human VAV3varl mRNA sequence is selected from SEQ ID NO: 1. The human VAV3var2 mRNA sequence is preferably selected from one of SEQ ID NO: 2, NCBI RefSeq database entry NM_001079874.1, and single- nucleotide polymorphisms thereof, in particular the human VAV3var2 mRNA sequence is selected from SEQ ID NO: 2. The human VAV3varl protein sequence is preferably selected from one of SEQ ID NO: 3, NCBI RefSeq database entry NP_006104.4, and single- amino-acid mutations thereof, in particular the human VAV3varl protein sequence is selected from SEQ ID NO: 3. The human VAV3var2 protein sequence is preferably selected from one of SEQ ID NO: 4, NCBI RefSeq database entry NP_001073343.1, and single- amino-acid mutations thereof, in particular the human VAV3var2 portein sequence is selected from SEQ ID NO: 4. The above- mentioned National Center for Biotechnology Information (NCBI) database entry identifiers include the entry version number ".N"; and relate to NCBI RefSeq database release 66, 7 July 2014.
The person of skill appreciates that, for other animal spe¬ cies, the respective homologues of the above-mentioned proteins or mRNAs have to be used.
In the context of the present invention, "predicting" shall not be construed in an absolute sense, i.e. with the meaning, that with 100% certainty it can be predicted that a patient will definitely develop cancer or the cancer (of a patient having the cancer) has a certain outcome. Instead, the invention relates to asserting an increased risk of a patient for developing cancer or for having a certain outcome. Similarly, "diagnosing a can¬ cer" also shall not be construed in the sense that all cancer patients can be identified by the inventive method but instead in the sense that the patient may have a increased probability of having the cancer. Therefore, the inventive method can be a first lead to identify the cancer, possibly followed by more in¬ vasive tests.
The present invention is further exemplified by the following embodiments, yet without being limited thereto: Embodiment 1. Use of VAV3 protein, or of an isoform thereof, especially VAV3var2 protein, or of VAV3 mRNA, or of a transcript variant thereof, especially VAV3var2 mRNA, as a can¬ cer marker;
for determining whether to treat a patient with a chemotherapeu- tic compound based on a sample of a cancer from a patient, preferably wherein said chemotherapeutic compound is a platinum- based therapeutic compound, preferably cisplatin or carboplatin or an angiogenic inhibitor, preferably bevacizumab, or a taxane, preferably paclitaxel,
especially wherein said chemotherapeutic compound is a platinum- based therapeutic compound, preferably cisplatin or carboplatin; or
for diagnosing of a cancer in a tissue sample or in a body fluid sample, wherein VAV3var2 protein or VAV3var2 mRNA is used.
Embodiment 2. The use of embodiment 1, wherein said cancer is a solid cancer, preferably selected from the group of lym¬ phoma, breast cancer, prostate cancer, gastric cancer and ovar¬ ian cancer, more preferably epithelial ovarian cancer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
Embodiment 3. The use of embodiment 2, wherein said cancer is an epithelial ovarian cancer and is one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1.
Embodiment 4. The use of embodiment 1, wherein said cancer is leukaemia.
Embodiment 5. The use of any one of embodiments 1-4, wherein said cancer is a cancer prone to relapse, especially de¬ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
preferably an ovarian cancer prone to relapse, especially de¬ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
more preferably an ovarian cancer prone to relapse despite platinum-based therapy.
Embodiment 6. A method for diagnosing a cancer in a pa- tient, comprising:
A) providing a tissue sample or a body fluid sample of said pa¬ tient; and
B) measuring the amount of VAV3var2 protein or the amount of VAV3var2 mRNA in said sample; and
C) comparing said amount with a mean amount of VAV3var2 protein, or a mean amount of VAV3var2 mRNA, present in one or more sam¬ ples of known cancer state as a positive control, and/or with a mean amount of VAV3var2 protein, or a mean amount of VAV3var2 mRNA, present in one or more samples of known non-cancer state as a negative control; and
D)
diagnosing said patient as having said cancer if
-said measured amount is significantly higher than said mean amount of the negative control and/or,
-said measured amount is essentially equal to or signifi¬ cantly higher to said mean amount of the positive control; or diagnosing said patient as not having said cancer if
-said measured amount is significantly lower than or essen¬ tially equal to said mean amount of the negative control, and/or
-said measured amount is significantly lower than said mean amount of the positive control.
Embodiment 7. The method of embodiment 6, wherein said can¬ cer is a leukemia or a solid cancer, which solid cancer is pref¬ erably selected from the group of lymphoma, breast cancer, pros¬ tate cancer, gastric cancer and ovarian cancer, more preferably epithelial ovarian cancer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endo¬ metrioid epithelial ovarian cancer.
Embodiment 8. The method of embodiment 7, wherein said can¬ cer is an epithelial ovarian cancer and is one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1.
Embodiment 9. The method of any one of embodiments 6-8, wherein said cancer is a cancer prone to relapse, especially de¬ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
preferably an ovarian cancer prone to relapse, especially de¬ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof,
more preferably an ovarian cancer prone to relapse despite platinum-based therapy.
Embodiment 10. The use of the method of any one of embodi¬ ments 6-9, for predicting disease-free survival, event-free sur¬ vival, puncture-free survival and/or overall survival of said patient .
Embodiment 11. Use of an anti-VAV3var2 antibody or a frag¬ ment thereof for diagnosing a cancer in a patient or determining the state of a cancer patient with respect to cancer treatment, preferably in the method of any one of embodiments 6-10 or 15- 18, wherein the VAV3var2 protein amount is measured, and/or preferably wherein the antibody has an affinity to VAV3varl that is weaker than the antibody's affinity to VAV3var2, preferably at least 10 times weaker, more preferably at least 100 times weaker, especially at least 1000 times weaker.
Embodiment 12. A method to screen for candidate therapeutic compounds, especially for candidate compounds for cancer ther¬ apy, the method comprising
-providing at least one compound and
-measuring the binding affinity of the at least one compound to VAV3var2 protein or measuring the effect of the at least one compound on VAV3var2 protein or mRNA expression in a cell.
Embodiment 13. The method of embodiment 12, further compris¬ ing
-designating as a candidate therapeutic compound, especially as a candidate compound for cancer therapy, a compound of the at least one compounds, if said compound of the at least one com¬ pounds has a binding affinity of less than ΙΟΟΟηΜ, preferably less than ΙΟΟηΜ, more preferably less than ΙΟηΜ, especially less than InM to the VAV3var2 protein and/or if said compound of the at least one compounds significantly reduces VAV3var2 protein and/or VAV3var2 mRNA expression in the cell compared to a nega¬ tive control .
Embodiment 14. The method of embodiment 13, with the proviso that said designating is only performed if said compound of the at least one compounds has a binding affinity to the VAV3varl protein that is higher in numeric value than the binding affin¬ ity to the VAV3var2 protein, preferably 10 times higher, more preferably 100 times higher, especially 1000 times higher and/or if said compound of the at least one compounds has essentially no effect on expression of VAV3varl protein and/or VAV3varl mRNA expression in the cell.
Embodiment 15. A method of determining the state of a cancer patient with respect to cancer treatment, comprising
A) measuring the amount of VAV3 mRNA or protein expression in a cancer biopsy sample of the patient; and
B) comparing said amount with a mean amount of VAV3 protein, or a mean amount of VAV3 mRNA, present in one or more samples of known cancer state as a positive control, and/or with a mean amount of VAV3 protein, or a mean amount of VAV3 mRNA, present in one or more samples of known non-cancer state as a negative control; and
C)
designating the patient as being eligible for the treatment with a platinum-based chemotherapeutic compound, preferably cisplatin or carboplatin, or with an anti-angiogenic compound, preferably bevacizumab, or with a taxane, preferably paclitaxel, or combi¬ nations thereof, in particular designating the patient as being eligible for the treatment with a platinum-based chemotherapeu- tic compound, preferably cisplatin or carboplatin; if
-said measured amount is significantly lower than or essen¬ tially equal to said mean amount of the negative control, and/or
-if said measured amount is significantly lower than said mean amount of the positive control;
or designating the patient as being not eligible for the treat¬ ment with a platinum-based chemotherapeutic compound, preferably cisplatin or carboplatin, or with an anti-angiogenic compound, preferably bevacizumab, or with a taxane, preferably paclitaxel, or combinations thereof, in particular designating the patient as being eligible for the treatment with a platinum-based chemo- therapeutic compound, preferably cisplatin or carboplatin; if -said measured amount is significantly higher than said mean amount of the negative control, and/or
-said measured amount is essentially equal to or signifi¬ cantly higher to said mean amount of the positive control. Embodiment 16. The method of embodiment 15, wherein VAV3 is an isoform of VAV3, preferably VAV3var2.
Embodiment 17. The method of embodiments 15 or 16, wherein said cancer is a solid cancer, preferably selected from the group of lymphoma, breast cancer, prostate cancer, gastric can¬ cer and ovarian cancer, more preferably epithelial ovarian can¬ cer, even more preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
Embodiment 18. The method of embodiments 15 or 16, wherein said cancer is leukaemia.
The invention is further described by the following examples and the drawing figures, yet without being limited thereto.
Figure 1: CSC-Specific VAV3var2 Expression in Ovarian Cancer. Ovarian CSC were purified from the indicated cell lines using ABC drug transporter-dependent dye efflux (side population as¬ say) . The stem cell properties of the isolated cells (such as clonogenicity, tumorigenicity and asymmetric division) were con¬ firmed in functional assays. VAV3var2 expression in CSC and non- CSC (exhibiting neither dye efflux nor functional stem cell properties) was determined on transcript level using qRT-PCR analysis. Quantification was done by normalizing Ct to reference Ct (TBP) . A2780 is a commercially available human ovarian carci¬ noma cell line. A2780V is a variant cell line of A2780. IGROV1 is a commercially available human ovarian epithelial cancer cell line. TBP, TATA-box-binding protein. Ct, cycle threshold.
Figure 2: Selective upregulation of VAV3var2 in Ovarian Tu¬ mour Tissue. VAV3varl and VAV3var2 transcript level was deter¬ mined in ovarian tumor tissue (Tu, n=163) and healthy ovarian control tissue (N, n=34) using qRT-PCR. Quantification was done by normalizing Ct to reference Ct (TBP) . TBP, TATA-box-binding protein. Ct, cycle threshold.
Figure 3: VAV3 and VAV3var2 Predict Poor Outcome in Ovarian Cancer Patients. VAV3 (A), VAV3varl (B) and VAV3var2 (C) tran¬ script level was determined in tumor tissue from 147 ovarian cancer patients using qRT-PCR. The data were sub-divided into low- and high-expressing cases according to 50th percentile sta¬ tistics, and correlated to disease-free survival (left-hand pan- els) and overall survival (right-hand panels) . Statistical sig¬ nificance was determined using log-rank test.
Figure 4: VAV3var2, but not VAV3, predicts Disease-Free Sur¬ vival in Endometrioid Ovarian Cancer Cases. VAV3 (left-hand panel) and VAV3var2 (right-hand panel) transcript level was de¬ termined in tumor tissue from 25 endometrioid ovarian cancer pa¬ tients using qRT-PCR. The data were sub-divided into low- and high-expressing cases according to 50th percentile statistics, and correlated to disease-free survival. Statistical signifi¬ cance was determined using log-rank test.
Figure 5: This Western Blot demonstrates that, in contrast to leukocytes (Jurkat) where the longer VAV3 isoform, VAV3varl, is predominant, ovarian cancer cells (OVCAR-3) express truncated versions of VAV3 protein such as VAV3var2.
Figure 6: VAV3var2 levels are significantly higher in ovarian cancer patients refractory to standard platinum based therapy. VAV3var2 transcript levels were determined using qRT-PCR. The data were sub-divided into patients refractory (N=19), resistant (N=22) or sensitive (N=105) to platinum based therapy according to the GCIG consensus 2010. Quantification was done by normaliz¬ ing Ct to reference Ct (TBP) . TBP, TATA-box-binding protein. Ct, cycle threshold.
Figure 7: VAV3var2 expression predicts poor outcome in endo¬ metrial cancer patients. VAV3var2 transcript levels were deter¬ mined using qRT-PCR. VAV3var2 transcription is significantly higher in tumor tissue from 90 endometrial cancer patients than in healthy tissue (N=26) (left panel) . Quantification was done by normalizing Ct to reference Ct (TBP) . TBP, TATA-box-binding protein. Ct, cycle threshold. The data were sub-divided into low- and high-expressing cases according to 75th percentile sta¬ tistics, and correlated to overall survival (right panel) .Example 1
Results :
Using global mRNA profiling (gene array analysis) , it was found that total VAV3 (comprising VAV3varl and VAV3var2) was selec¬ tively overexpressed in ovarian cancer stem cells (CSC) recov¬ ered from ovarian cancer cell lines. Subsequent qRT-PCR analysis confirmed this result, and further showed that of the two vari- ants only VAV3var2 was overexpressed in ovarian CSC (Figure 1) . qRT-PCR-based analysis of VAV3 isoform expression in ovarian tu¬ mor tissue (n=163) and healthy control tissue (n=34) subse¬ quently revealed strong upregulation of VAV3var2 in tumor tis¬ sue, whereas VAV3varl was not upregulated in tumor tissue (Fig¬ ure 2) . Thus, it was shown for the first time that tumoral VAV3 expression is almost exclusively due to expression of VAV3var2. Importantly, multivariate analyses showed that high expression of total VAV3 (Figure 3A) and VAV3var2 (Figure 3C) , but not of VAV3varl (Figure 3B) , statistically significantly correlated with poor disease-free survival (DFS) and poor overall survival (OS) . Furthermore, sub-classification of the patient cohort into major ovarian cancer subtypes (i.e., serous, mucinous, endo¬ metrioid) revealed that VAV3var2 was a robust predictor of pa¬ tient outcome throughout the various subtypes (DFS: serous, p<0.001; mucinous, p=0.025; endometrioid, p=0.013). In contrast, total VAV3 statistically significantly predicted DFS only in the serous (p=0.002) and the mucinous (p=0.022) subgroups, but not in the endometrioid subgroup (p=0.128) (Figure 4) . Thus, the re¬ sults show that total VAV3 and, in particular, VAV3var2 as novel, potentially CSC-specific, biomarkers in the principal subtypes of ovarian cancer. These findings represent the ration¬ ale for therapeutic targeting of total VAV3 and, in particular, VAV3var2 in cancer, especially ovarian cancer and/or ovarian CSC. Moreover, it was found that platinum-refractory patients exhibited significantly higher levels of total VAV3 and VAV3var2 than non-platinum-refractory patients. In addition, total VAV3 and VAV3var2 were significantly higher in patients undergoing relapse than in patients not undergoing relapse.
146 of the 147 ovarian cancer patients mentioned previously were sub-divided into patients refractory (N=19), resistant (N=22) or sensitive (N=105) to platinum based therapy according to the Gy- necoligical Cancer InterGroup (GCIG) consensus 2010. VAV3var2 levels are significantly higher in Ovarian Cancer Patients re¬ fractory to standard platinum based therapy (see Fig. 6) .
Therefore, the data show total VAV3, and in particular, VAV3var2 as predictors of platinum refractoriness and cancer re¬ currence in ovarian cancer.
Materials and Methods: Assay of CSC-specific VAV3var2 expression in ovarian cancer lines (Fig. 1) . CSCs were isolated by FACS using the side popu¬ lation assay (see e.g. Boesch et al, 2014) . The stem cell prop¬ erties of the isolated cells (such as clonogenicity, tumori- genicity and asymmetric division) were confirmed in functional assays. VAV3var2 expression in CSC and non-CSC (exhibiting nei¬ ther dye efflux nor functional stem cell properties) was deter¬ mined on transcript level using qRT-PCR analysis. Quantification was done by normalizing Ct to reference Ct (TBP) . RNA extrac¬ tion, reverse transcription and qRT-PCR was performed as de¬ scribed below.
Assay of CSC-specific VAV3var2 expression in ovarian cancer bi¬ opsies (Fig. 2) . Tumor tissue (Tu) and healthy control tissue (N) was obtained from patients undergoing debulking surgery. Tissue was flash-frozen and pulverized. RNA extraction, reverse transcription and qRT-PCR was performed as described below.
VAV3var2 and cancer outcome (Figs. 3 and 4) . Tumor tissue was obtained from patients undergoing debulking surgery. Tissue was flash-frozen and pulverized. RNA extraction, reverse transcrip¬ tion and qRT-PCR was performed as described below. 50th percen¬ tile statistics was used to divide the cohort into low- and high-expressing cases, and DFS and OS were analysed using the Kaplan-Meier method. A total of 147 ovarian cancer patients were included in this study with a median age of 63 at diagnosis. 72% were in advanced FIGO stages 3/4; roughly 50% had serous, 30% mucinous and 17% endometrioid subtypes.
VAV3 isoform expression in lymphocytes and ovarian cancer cells (Fig. 5) : Protein fractions were harvested from ovarian cancer cells using a lysis buffer containing NP-40 as detergent and protease inhibitors for prevention of protein degradation. 25 pg of denatured protein were loaded per slot in reducing sample buffer (purchased from Life Technologies) and separated for 90 min at 150 V on a prefabricated SDS-PAGE gel (NuPAGE; purchased from Life Technolgies) . MOPS SDS was used as running buffer (purchased from Life Technologies) . Separated protein fractions were blotted onto a standard nitrocellulose membrane (120 min at 30 V) . The nitrocellulose membrane was washed in TTBS and incu¬ bated for 60 min in 1% (w/v) milk powder to block unspecific binding sites. After washing, blotted proteins were incubated overnight with a rabbit anti-human VAV3 antibody (clone EP1130Y; purchased from Novus Biologicals) . The next day, the membrane was washed in TTBS and incubated for 60 min with a HRP-linked polyclonal anti-rabbit antibody (purchased from Cell Signaling) . Proteins were finally detected by enhanced chemiluminescence us¬ ing commercially available reagents (LumiGlo/Peroxid Reagent; purchased from Cell Signaling) . Alpha-tubulin was used as load¬ ing control .
This Western Blot demonstrates that, in contrast to leuko¬ cytes (Jurkat cells) where the longer VAV3varl is predominant, ovarian cancer cells express truncated versions of VAV3 protein such as VAV3var2.
Real-Time PCR:
For qRT-PCR-based expression profiling, 2*106 cells were washed twice in ice-cold PBS and pelleted in 1.5 ml Eppendorf tubes (centrifugation for 3 min at 250 g at 4°C) . The super¬ natant was discarded and the pellet was immediately frozen at - 80°C.
Cell preparation: This relates to cultured cells from estab¬ lished cancer cell lines. A2780 cells were purchased from Sigma- Aldrich, Vienna, Austria. OVCAR-3 cells were purchased from ATCC, Wesel, Germany. IGROV1 cells were kindly obtained from Prof. R. Brown, London, UK, and the A2780 variant cell line A2780V was generously provided by Prof. R. Zeillinger, Vienna, Cells were maintained in appropriate culture medium at 37° in a humidified atmosphere containing 5% C02. Cells were harvested using trypsin and prepared for qRT-PCR as described above.
RNA Isolation
For isolation of RNA, cell pellet was resuspended in 1 ml TRI Reagent®, vortexed and incubated for 5 min at RT . 200 μΐ chloro¬ form were added and the mixture was thoroughly vortexed for 15 sec, followed by incubation for 15 min at RT . After centrifuga- tion for 10 min at 12, 000 g at 4°C, the upper, aqueous phase (containing RNA) was transferred to another Eppendorf tube. 500 μΐ isopropyl alcohol were added to the RNA solution, followed by gentle vortexing for 10 sec and incubation for 10 min at RT . RNA was spun down for 8 min at 12, 000 g at 4°C and the supernatant was removed. 1 ml ethanol (70%) was added to the tube and the RNA was again pelleted (centrifugation for 5 min at 12, 000 g at 4°C) . The supernatant was discarded and the pellet was dried for 15 min under laminar flow conditions. Dried RNA pellet was re- suspended in 50 μΐ nuclease-free water and immediately placed on ice. RNA concentration was determined using NanoDrop™ Lite.
Reverse Transcription
Reverse transcription of RNA was accomplished using Moloney Murine Leukaemia Virus (M-MLV) reverse transcriptase and random hexamer priming. Experimentally, two separate mixes were pre¬ pared :
Mix I :
Mix I was incubated for 5 min at 65°C and immediately chilled on ice. Mix II was added to Mix I, yielding a final reaction volume of 40 μΐ . The reaction was performed as follows: 10 min at 25°C (primer annealing), 60 min at 37°C (reverse transcrip¬ tion), 15 min at 70°C (heat inactivation) , and hold at 4°C.
Real-Time PCR
qRT-PCR of cDNA templates was done using the ABI PRISM® 7900HT Sequence Detection System and corresponding software (SDS 2.3). Amplification products were detected using fluorescently- labeled oligonucleotide hybridization probes (FAM/BHQ, FAM/TAMRA) . Relative quantification of gene expression was done by normalizing the respective Ct values to reference Ct value (TBP) . The reaction mix contained the following components:
25 μΐ PCR Mix:
Component Volume [μΙ] Final Concentration
Aqua destillata 2,375
cDNA template (10 ng/μΙ) 5 2 ng/μΙ
Primer F (10 pM) 2,25 900 nM Primer R (10 pM) 2,25 900 nM
Probe (10 pM) 0,625 250 nM
TaqMan Universal PCR Master Mix (2x) 12,5 1 x
The reaction was performed as follows: 2 min at 50°C, 10 min at 95° C (denaturing) , and 45 amplification cycles comprising 15 sec at 95°C (denaturing) and 1 min at 60°C (primer annealing and
DNA synthesis ) , respectively .
Primer Sequences
Transcript , Primer/Probe , Symbol SEQ Sequence D/Q
TBP Sense TBP_s(1)_F 5 CAC GAA CCA CGG CAC TGA TT
Antisense TBP_as(1)_F 6 TTT TCT TGC TGC CAG TCT GGA C
Taq TBP Taq(1)_E 7 TGT GCA CAG GAG CCA AGA GTG AAG A FAM/BHQ
VAV3 Sense VAV3ALL_s(1)_A 8 CCA GAG AAA CGG ACC AAT GG
Antisense VAV3ALL_as(1)_A 9 CCT AAT GAC CTG CAT CTT TGG TAA
Taq VAV3ALL_Taq(1)_A 10 CTG CGA AGA ACT CCT AAA CAG GTG GAT CC FAM/TAMRA
VAV3var1+2 Sense VAV3var1 Ex18b_s(1)_A 11 AGA GTT AAT TCT GGT GAA CAA GGG A
Sense VAV3var2Ex18a_s(1 )_A 12 TTA CAT TTC TTT CAG AAC AAG GGA CA
Antisense VAV3var1+2_as(1)_A 13 TCC ACC TGT TTA GGA GTT CTT CG
Taq VAV3var1 +2 Taq(1) A 14 TCA AAC TAC CAG AGA AAC GGA CCA ATG GAC FAM/TAMRA
SEQ: SEQ ID NO, D/Q: Dye/Quencher.
Example 2
Tumor tissue and, where possible, healthy adjoining tissue was obtained from endometrial cancer patients undergoing debulking surgery. Tissue was flash-frozen and pulverized. RNA extraction, reverse transcription and qRT-PCR was performed as described in Example 1. 75th percentile statistics was used to divide the co¬ hort into low- and high-expressing cases, and overall survival was analysed using the Kaplan-Meier method. A total of 90 endo¬ metrial cancer patients were included in this study. VAV3var2 expression predicts poor outcome in endometrial cancer patients, as shown in Fig. 7.
References in the non-patent literature
Armstrong, Deborah K., et al . "Intraperitoneal cisplatin and paclitaxel in ovarian cancer." New England Journal of Medicine 354.1 (2006) : 34-43.
Boesch, Maximilian, et al . "The side population of ovarian cancer cells defines a heterogeneous compartment exhibiting stem cell characteristics." Oncotarget 5 (2014).
Brantley-Sieders , Dana M., et al . "Host deficiency in Vav2/3 guanine nucleotide exchange factors impairs tumor growth, sur¬ vival, and angiogenesis in vivo." Molecular Cancer Research 7.5 (2009) : 615-623.
Chang, K. H. et al . Vav3 collaborates with pl90-BCR-ABL in lymphoid progenitor leukemogenesis , proliferation, and survival. Blood 120, 800-811, doi : 10.1182/blood-2011-06-361709 (2012)
Golebiewska, Anna, et al . "Critical appraisal of the side population assay in stem cell and cancer stem cell research." Cell Stem Cell 8.2 (2011): 136-147.
Hennessy, Bryan T . , Robert L. Coleman, and Maurie Markman. "Ovarian cancer." The Lancet 374.9698 (2009): 1371-1382.
Hunter, Sonja G., et al . "Essential role of Vav family gua¬ nine nucleotide exchange factors in EphA receptor-mediated an- giogenesis." Molecular and cellular biology 26.13 (2006): 4830- 4842.
Lin, K. Y. et al . (A) Clinical significance of increased gua¬ nine nucleotide exchange factor Vav3 expression in human gastric cancer. Molecular cancer research : MCR 10, 750-759, doi : 10.1158/1541-7786.MCR-11-0598-T (2012)
Lin, K. T. et al . (B) Vav3-racl signaling regulates prostate cancer metastasis with elevated Vav3 expression correlating with prostate cancer progression and posttreatment recurrence. Cancer Res 72, 3000-3009, doi : 10.1158/0008-5472. CAN-11-2502 (2012).
Macarron, Ricardo, et al . "Impact of high-throughput screen¬ ing in biomedical research." Nature reviews Drug discovery 10.3 (2011) : 188-195.
Mayr, Lorenz M., and Dejan Bojanic. "Novel trends in high- throughput screening." Current opinion in pharmacology 9.5 (2009) : 580-588.
Medema, Jan Paul. "Cancer stem cells: the challenges ahead." Nature cell biology 15.4 (2013): 338-344.
Pattabiraman, Diwakar R., and Robert A. Weinberg. "Tackling the cancer stem cells—what challenges do they pose?." Nature Re¬ views Drug Discovery 13.7 (2014) : 497-512.
Peccorelli et al . "Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium." International Journal of Gyne¬ cology & Obstetrics 105.2 (2009): 103-104.
Perren, Timothy J., et al . "A phase 3 trial of bevacizumab in ovarian cancer." New England Journal of Medicine 365.26 (2011): 2484-2496.
Qi, Hongyi, et al . "Proteomic characterization of the cellu¬ lar response to chemopreventive triterpenoid astragaloside IV in human hepatocellular carcinoma cell line HepG2. " International journal of oncology 36.3 (2010): 725-735.
Sundberg, Steven A. "High-throughput and ultra-high- throughput screening: solution-and cell-based approaches." Cur¬ rent opinion in biotechnology 11.1 (2000): 47-53.
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Claims

Claims :
1. Use of VAV3var2 protein or of VAV3var2 mRNA as a cancer marker;
for determining whether to treat a patient with a chemotherapeu- tic compound based on a sample of a cancer from a patient; or for diagnosing of a cancer in a tissue sample or in a body fluid sample .
2. The use of claim 1, wherein said cancer is a solid cancer.
3. The use of claim 2, wherein said cancer is selected from the group of ovarian cancer, endometrial cancer, lymphoma, breast cancer, prostate cancer and gastric cancer.
4. The use of claim 3, wherein said cancer is an epithelial ovarian cancer, preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
5. The use of claim 4, wherein said cancer is one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1.
6. The use of claim 1, wherein said cancer is leukaemia.
7. The use of any one of claims 1-6, wherein the use is for de¬ termining whether to treat a patient with a chemotherapeutic compound based on a sample of a cancer from a patient.
8. The use of claim 7, wherein said chemotherapeutic compound is a platinum-based therapeutic compound or an angiogenic in¬ hibitor or a taxane or combinations thereof.
9. The use of claim 8, wherein the chemotherapeutic compound is selected from the group of cisplatin, carboplatin, bevacizumab, paclitaxel and combinations thereof.
10. The use of any one of claims 1-9, wherein said cancer is a cancer prone to relapse, especially despite platinum-based, tax- ane-based or anti-angiogenic therapy, or combinations thereof, preferably an ovarian cancer prone to relapse, especially de¬ spite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof.
11. A method for diagnosing a cancer in a patient, comprising:
A) providing a tissue sample or a body fluid sample of said pa¬ tient; and
B) measuring the amount of VAV3var2 protein or the amount of VAV3var2 mRNA in said sample; and
C) comparing said amount with a mean amount of VAV3var2 protein, or a mean amount of VAV3var2 mRNA, present in one or more sam¬ ples of known cancer state as a positive control, and/or with a mean amount of VAV3var2 protein, or a mean amount of VAV3var2 mRNA, present in one or more samples of known non-cancer state as a negative control; and
D)
diagnosing said patient as having said cancer if
-said measured amount is significantly higher than said mean amount of the negative control and/or,
-said measured amount is essentially equal to or signifi¬ cantly higher to said mean amount of the positive control; or diagnosing said patient as not having said cancer if
-said measured amount is significantly lower than or essen¬ tially equal to said mean amount of the negative control, and/or
-said measured amount is significantly lower than said mean amount of the positive control.
12. The method of claim 11, wherein said cancer is a solid can¬ cer .
13. The method of claim 12, wherein the cancer is selected from the group of ovarian cancer, endometrial cancer, lymphoma, breast cancer, prostate cancer and gastric cancer.
14. The method of claim 13, wherein the cancer is epithelial ovarian cancer, preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
15. The method of claim 14, wherein said cancer is one of stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, any grade of stage IC, any grade of stage II, any grade of stage III or any grade of stage IV, preferably stage IA/B grade 1, stage IA/B grade 2, stage IA/B grade 3, more preferably stage IA/B grade 1 or stage IA/B grade 2, especially stage IA/B grade 1.
16. The method of any one of claims 11-15, wherein said cancer is a cancer prone to relapse, especially despite platinum-based, taxane-based or anti-angiogenic therapy, or combinations
thereof, preferably an ovarian cancer prone to relapse, espe¬ cially despite platinum-based, taxane-based or anti-angiogenic therapy, or combinations thereof.
17. The method of claim 11, wherein the cancer is a leukemia.
18. The use of the method of any one of claims 11-17, for pre¬ dicting disease-free survival, event-free survival, puncture- free survival and/or overall survival of said patient.
19. Use of an anti-VAV3var2 antibody or a fragment thereof for diagnosing a cancer in a patient or determining the state of a cancer patient with respect to cancer treatment, preferably in the method of any one of claims 6-10 or 15-18, wherein the VAV3var2 protein amount is measured, and/or
preferably wherein the antibody has an affinity to VAV3varl that is weaker than the antibody's affinity to VAV3var2, preferably at least 10 times weaker, more preferably at least 100 times weaker, especially at least 1000 times weaker.
20. A method to screen for candidate therapeutic compounds, es¬ pecially for candidate compounds for cancer therapy, the method comprising
-providing at least one compound and
-measuring the binding affinity of the at least one compound to VAV3var2 protein or measuring the effect of the at least one compound on VAV3var2 protein or mRNA expression in a cell, wherein the method is a high-throughput screening method.
21. The method of claim 20, further comprising
-designating as a candidate therapeutic compound, especially as a candidate compound for cancer therapy, a compound of the at least one compounds, if said compound of the at least one com¬ pounds has a binding affinity of less than ΙΟΟΟηΜ, preferably less than ΙΟΟηΜ, more preferably less than ΙΟηΜ, especially less than InM to the VAV3var2 protein and/or if said compound of the at least one compounds significantly reduces VAV3var2 protein and/or VAV3var2 mRNA expression in the cell compared to a nega¬ tive control .
22. The method of claim 21, with the proviso that said designat¬ ing is only performed if said compound of the at least one com¬ pounds has a binding affinity to the VAV3varl protein that is higher in numeric value than the binding affinity to the
VAV3var2 protein, preferably 10 times higher, more preferably 100 times higher, especially 1000 times higher and/or if said compound of the at least one compounds has essentially no effect on expression of VAV3varl protein and/or VAV3varl mRNA expres¬ sion in the cell.
23. A method of determining the state of a cancer patient with respect to cancer treatment, comprising
A) measuring the amount of VAV3 mRNA or protein expression in a cancer biopsy sample of the patient; and
B) comparing said amount with a mean amount of VAV3 protein, or a mean amount of VAV3 mRNA, present in one or more samples of known cancer state as a positive control, and/or with a mean amount of VAV3 protein, or a mean amount of VAV3 mRNA, present in one or more samples of known non-cancer state as a negative control; and
C)
designating the patient as being eligible for the treatment with a platinum-based chemotherapeutic compound, preferably cisplatin or carboplatin, or with an anti-angiogenic compound, preferably bevacizumab, or with a taxane, preferably paclitaxel, or combi¬ nations thereof, if
-said measured amount is significantly lower than or essen¬ tially equal to said mean amount of the negative control, and/or
-if said measured amount is significantly lower than said mean amount of the positive control; or designating the patient as being not eligible for the treat¬ ment with a platinum-based chemotherapeutic compound, preferably cisplatin or carboplatin, or with an anti-angiogenic compound, preferably bevacizumab, or with a taxane, preferably paclitaxel, or combinations thereof, if
-said measured amount is significantly higher than said mean amount of the negative control, and/or
-said measured amount is essentially equal to or signifi¬ cantly higher to said mean amount of the positive control;
24. The method of claim 23, wherein the VAV3 mRNA or VAV3 pro¬ tein is VAV3var2 mRNA or VAV3var2 protein, respectively.
25. The method of claim 23 or 24, wherein said cancer is a solid cancer .
26. The method of claim 25, wherein said cancer is selected from the group of ovarian cancer, endometrial cancer, lymphoma, breast cancer, prostate cancer and gastric cancer.
27. The method of claim 26, wherein said cancer is epithelial ovarian cancer, preferably serous, mucinous or endometrioid epithelial ovarian cancer, especially endometrioid epithelial ovarian cancer.
28. The method of claims 23 or 24, wherein said cancer is leu¬ kaemia .
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