WO2015011481A1 - Biomarqueurs - Google Patents

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WO2015011481A1
WO2015011481A1 PCT/GB2014/052268 GB2014052268W WO2015011481A1 WO 2015011481 A1 WO2015011481 A1 WO 2015011481A1 GB 2014052268 W GB2014052268 W GB 2014052268W WO 2015011481 A1 WO2015011481 A1 WO 2015011481A1
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subject
sample
cancer
biomarker
biomarkers
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PCT/GB2014/052268
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English (en)
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Roz BANK
David Cairns
Alexandre ZOUGMAN
Fiona COLLINSON
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Cancer Research Technology Limited
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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
    • G01N33/57484—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
    • G01N33/57488—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 involving compounds identifable in body fluids
    • 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

Definitions

  • the present invention relates generally to the field of cancer diagnosis and treatment.
  • the invention relates to methods for determining whether subjects will respond to an anti-cancer therapy.
  • the invention is particularly applicable for identifying patients who will respond to administration of an anti-angiogenic agent such as bevacizumab in the treatment of ovarian and other cancers.
  • Cancer is one of the leading causes of death and represents a major socioeconomic cost in most developed societies. Whilst recent advances in diagnosis and treatment have led to increases in survival rates for some cancers, effective management of this disease is still one of the greatest challenges facing many healthcare systems. In particular, newer antibody-based therapeutic strategies are now able to target more specifically the underlying pathology of the disease, but are typically more expensive than traditional treatments and may not be effective in many subjects. There is therefore an urgent need for methods which allow the stratification of patients according to their likely responsiveness to anti-cancer therapies.
  • OC epithelial ovarian cancer
  • OC causes significant morbidity and mortality worldwide with an estimated 21,880 new cases and 13,850 attributable deaths in 2010 (1).
  • Standard treatment for many years has involved debulking surgery followed by systemic platinum-based chemotherapy (2).
  • VEGF vascular endothelial growth factor
  • VEGF is involved in stimulating angiogenesis and may therefore be critical to tumour growth.
  • ICON7 is a two-arm phase III international randomised open-label trial of 1528 women with high risk early-stage or advanced-stage ovarian cancer, comparing six cycles of standard chemotherapy (carboplatin and paclitaxel) to six cycles of chemotherapy plus the addition of concurrent and maintenance bevacizumab (7.5 mg/kg every 3 weeks; Hoffmann-La Roche, Switzerland), up to a maximum of 18 cycles in total.
  • the results of the trial demonstrated that the addition of bevacizumab improved progression-free survival (PFS) in women with ovarian cancer, but although statistically significant, over the entire trial population the PFS benefit was only 1.5 months (5).
  • PFS progression-free survival
  • biomarkers which are indicative of the responsiveness of a subject suffering from cancer to an anti-angiogenic therapy.
  • biomarkers which can be used to predict the effectiveness of bevacizumab in the treatment of ovarian cancer.
  • the present invention provides a method of predicting responsiveness of a subject to an anti-angiogenic agent for treating cancer, the method comprising: (i) determining a level of two or more biomarkers in a sample from the subject, wherein the biomarkers are selected from the group consisting of ⁇ -acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and cancer antigen 125 (CA-125); and (ii) comparing the levels of the biomarkers in the sample to predetermined reference values; wherein the level of the biomarkers in the sample compared to the predetermined reference values is indicative of responsiveness of the subject to the anti-angiogenic agent for treating cancer.
  • AGP ⁇ -acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • CA-125 cancer antigen 125
  • the anti-angiogenic agent comprises an antibody which binds to vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the antibody comprises bevacizumab.
  • the cancer is selected from: ovarian, colorectal, renal and glioblastoma multiforme cancer.
  • the subject is a human.
  • the sample comprises whole blood, serum or plasma.
  • each predetermined reference value comprises a cut-off value for the level of the biomarker in the sample, biomarker levels above or below the cut-off value being differentially associated with responsiveness to the anti-angiogenic agent.
  • the method comprises determining three or four of said biomarkers in the sample from the subject.
  • an increased level of ⁇ -acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4) and/or mesothelin in the sample compared to the predetermined reference values is indicative of increased responsiveness to the anti-angiogenic agent.
  • AGP ⁇ -acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin in the sample compared to the predetermined reference values is indicative of increased responsiveness to the anti-angiogenic agent.
  • a decreased level of cancer antigen 125 (CA-125) in the sample compared to the predetermined reference value is indicative of increased responsiveness to the anti- angiogenic agent.
  • the present invention provides a method of treating cancer in a subject, the method comprising (i) predicting responsiveness of the subject to an anti-angiogenic agent by a method as defined above; (ii) treating the subject with an anti-angiogenic agent if the subject is predicted to be responsive thereto.
  • the anti-angiogenic agent comprises an antibody which binds to vascular endothelial growth factor (VEGF).
  • VEGF vascular endothelial growth factor
  • the antibody comprises bevacizumab.
  • the method further comprises treating the subject with chemotherapy. If the subject is predicted to be non-responsive to bevacizumab, the subject is preferably treated with chemotherapy alone.
  • the chemotherapy comprises administration of carboplatin and/or paclitaxel to the subject.
  • the present invention provides an anti-angiogenic agent for use in treating cancer in a subject, wherein the subject has a level of two or more biomarkers selected from the group consisting of ⁇ -acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and CA- 125 which is indicative of responsiveness of the subject to the anti-angiogenic agent.
  • AGP ⁇ -acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin mesothelin
  • CA- 125 which is indicative of responsiveness of the subject to the anti-angiogenic agent.
  • the anti-angiogenic agent comprises bevacizumab.
  • the subject has a decreased level of at least 2 of said biomarkers compared to predetermined reference values.
  • the subject has a decreased level of 3 or more of said biomarkers compared to the predetermined reference values.
  • the subject has been predicted to be responsive to the anti-angiogenic agent by a method as defined above.
  • the biomarker levels are determined in whole blood, serum or plasma.
  • the present invention provides a pharmaceutical combination comprising an anti-angiogenic agent and a chemotherapeutic agent for simultaneous, separate or sequential use in treating cancer in a subject, wherein the subject has a level of two or more biomarkers selected from the group consisting of al-acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and CA-125 which is indicative of responsiveness of the subject to the anti-angiogenic agent.
  • the anti-angiogenic agent comprises bevacizumab.
  • the chemotherapeutic agent comprises carboplatin and/or paclitaxel.
  • the present invention provides a kit for predicting responsiveness of a subject to an anti-angiogenic agent for treating cancer, the kit comprising reagents suitable for detecting two or more biomarkers in a sample from the subject, wherein the biomarkers are selected from the group consisting of al-acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and CA- 125.
  • AGP al-acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin CA- 125.
  • the kit comprises antibodies which bind to two or more of said biomarkers.
  • the kit comprises an ELISA assay for two or more of said biomarkers.
  • the present invention provides a method for predicting progression and/or relapse of ovarian cancer in a subject, the method comprising (i) determining a level of one or more biomarkers in a sample from the subject, wherein the biomarker is selected from the group consisting of al-acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and cancer antigen 125 (CA-125); and (ii) comparing the level of the biomarker in the sample to a predetermined reference value; wherein the level of the biomarker in the sample compared to the predetermined reference value is indicative of the progression of ovarian cancer in the subject.
  • AGP al-acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • CA-125 cancer antigen 125
  • the level of the biomarker in the sample compared to the predetermined reference value is indicative of progression-free survival time of the subject.
  • the biomarker is al-acid glycoprotein (AGP). In an alternative embodiment, the biomarker is fms-like tyrosine kinase-4 (FLT4). In another embodiment, the biomarker is mesothelin.
  • the present invention provides a method for predicting progression and/or relapse of ovarian cancer in a subject, the method comprising: (i) determining a level of one or more biomarkers in a sample from the subject, wherein the biomarker is selected from the group consisting of otl-acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and cancer antigen 125 (CA-125); and
  • AGP otl-acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • CA-125 cancer antigen 125
  • the level of the biomarker in the sample compared to the predetermined reference value may be indicative of progression-free survival time of the subject.
  • the biomarker may be otl-acid glycoprotein (AGP).
  • the biomarker may be fms-like tyrosine kinase-4 (FLT4).
  • the biomarker may be mesothelin.
  • the present invention provides a method of predicting responsiveness of a subject to a chemotherapeutic agent for treating cancer, the method comprising:
  • biomarkers are selected from the group consisting of ⁇ -acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4), mesothelin and cancer antigen 125 (CA-125); and
  • AGP ⁇ -acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • CA-125 cancer antigen 125
  • the chemotherapeutic agent may be a platinum-based chemotherapeutic agent.
  • the chemotherapeutic agent may be selected from the following: cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, lipoplatin.
  • the cancer may be selected from: ovarian, colorectal, renal and glioblastoma multiforme cancer.
  • the subject may be a human.
  • the method may involve determining two, three or four of said biomarkers in the sample from the subject.
  • the sample may comprise whole blood, serum or plasma.
  • Each predetermined reference value may comprise a cut-off value for the level of the biomarker in the sample, biomarker levels above or below the cut-off value being differentially associated with responsiveness to the chemotherapeutic agent.
  • the predetermined reference values may comprise values within one or more of the following ranges:
  • the present invention provides a method of treating cancer in a subject, the method comprising:
  • the present invention provides a method of treating cancer in a subject, the method comprising:
  • Figure 1 Timing of serum samples as part of the ICON7 trial.
  • samples were taken pre-cycle 1 (1 and 2), post- cycle 1 (3), pre-cycles 2 (4) and 6 (5), post-cycle 6 (6) and at 3, 6 and 9 months from completion of chemotherapy (7-9) and at either 5 years from randomisation or at progression (whichever occurred first) (10).
  • samples were taken pre-cycle 1 (1), pre cycles 2 and 6 (4 and 5) and at either 5 years from randomisation or at progression (whichever occurred first) (10).
  • Figure 2 Relationship between mesothelin and response to treatment and the kinetics of change in FLT4 related to treatment, (a) Longitudinal changes in mesothelin between time-points 1 and 4 in responders and non-responders in the discovery cohort as determined confirmed by ELISA. Longitudinal changes in mesothelin between time-points 1 and 4 in early and late progressors in the validation cohort in the standard arm (b)[i] and experimental arm (b)[ii] measured by immunoassay. Data points are the mean with dotted lines representing ⁇ standard error, (c) Relationship between mesothelin and response to treatment by risk of progression status in validation cohort.
  • Longitudinal box plots* showing changes in mesothelin concentration across the sample time-points in early and late progressors in the standard and experimental arms for patients at high risk of progression and patients not at high risk of progression (high risk of progression defined as FIGO stage IV disease or FIGO stage III disease AND >1.0cm residual disease after debulking surgery), (d) Longitudinal box plots* showing changes in FLT4 median concentration over time-points in ICON7 sampling. Panels show early and late progressors in terms of PFS in standard and experimental arms of trial.
  • FIG. 3 Predictive potential of biomarker index in signature-negative and signature-positive patient groups.
  • Graphs show mean+/-standard error of the mean (SEM). For AGP the immunoassay measured both AGP1 and 2.
  • Figure 5 Progression-free survival in validation cohort I.
  • Figure 7 Longitudinal patterns in AGP concentration, (a) Longitudinal box plots* showing changes in AGP median concentration over time-points in ICON7 sampling scheme. Panels show early and late progressors in the standard and experimental arms of trial and (b) longitudinal patterns in AGP by risk of progression status (as above but split by risk of progression, defined as patients with FIGO stage IV disease OR FIGO stage III disease AND >1.0 cm residual disease after debulking surgery).
  • Figure 8. Prognostic and predictive potential of candidate biomarkers validation cohort I. Kaplan- Meier estimates of survival functions for biomarkers at baseline (time-point 1) dichotomized at optimum cut-point in all patients (column a), standard arm (column b), experimental arm (column c) and for all patients with interaction for treatment group included in the model (column d). Chi- squared results and associated p-values from log-rank tests to compare survival curves shown on all plots. Hazard ratios for interaction term for predictive ability and associated p-value is also displayed in column d.
  • Figure 9 Baseline concentrations of mesothelin, FLT4, AGP and CA125 in: a), validation cohort I, b). validation cohort II, and c). both validation cohorts combined. Values are shown for patients in each arm of the trial (CP or CP plus bevacizumab) and subdivided into whether low or high risk.
  • the solid red lines show the median values for each and the dashed red lines show the upper limit of the normal range, as described in the Examples below, for each analyte. In the case of AGP the upper limit of normal for both females>50 years of age (upper line) and ⁇ 50 years are shown (lower line).
  • the present invention advantageously provides a method for predicting responsiveness of a subject to an anti-angiogenic therapy for cancer.
  • the inventors have determined that surprisingly, particular combinations of biomarkers are predictive of therapeutic efficacy, whereas individual biomarkers may be only of prognostic value in ovarian cancer.
  • the present invention demonstrates that responsiveness to anti-angiogenic agents such as bevacizumab is not simply associated with prognosis (e.g. risk of disease progression) as such.
  • responsive subjects show a specific molecular profile which can be used to target anti-angiogenic therapy more effectively to those patients which will benefit, thereby improving clinical outcomes and providing a more efficient use of resources. Predicting Responsiveness
  • the present method relates to predicting responsiveness of a subject to an anti-angiogenic agent.
  • the method may be used to determine whether or not administration of an anti-angiogenic therapy to the subject is likely to provide a clinical benefit.
  • the benefit may be, for example, in terms of a remission of the disease, an alleviation of one or more symptoms of the disease, a delay in progression of disease or an increase in survival time.
  • responsiveness of the subject and/or clinical benefit may be measured in terms of an increase in progression-free survival (PFS) time following treatment with the anti-angiogenic agent (e.g. bevacizumab).
  • PFS progression-free survival
  • the biomarkers described herein may be used in prognostic applications, e.g. for providing an indication of disease status (as opposed to predicting responsiveness to therapy, as discussed in the preceding paragraph).
  • the invention provides methods for predicting progression of cancer (e.g. ovarian cancer) comprising detecting biomarkers as described herein.
  • the method may be used to predict progression-free survival time of the subject.
  • the method may be used to predict severity of the disease, e.g. the degree to which the cancer is advanced or the rate at which the cancer is predicted to advance in the future.
  • prognostic methods may be performed in a similar manner to the methods described herein for predicting responsiveness to therapy, e.g. on the same types of subjects, using similar biomarker detection methods, reference values and on the same types of sample.
  • the methods may also be used in combinations as described below.
  • the subject is a human.
  • the method of the present invention is not limited to humans, and may also be performed on e.g. non-human mammals.
  • the subject is an adult human, although in some embodiments the method may be performed on a child or infant.
  • the subject is female.
  • the subject is typically suffering from cancer, or suspected to be suffering from cancer.
  • the method may be used, for instance, to select a personalized treatment protocol for the subject.
  • the method may be performed before the subject has received (e.g. an anti-angiogenic) treatment for cancer, or after treatment has already commenced, for instance in order to decide whether to continue treating the subject with an anti-angiogenic therapy.
  • Anti-Angiogenic Agents are indicative of responsiveness to anti-angiogenic agents for treating cancer.
  • anti-angiogenic agent it is meant any therapy which prevents, reduces or restricts angiogenesis, i.e. the growth of new blood vessels, specifically in the treatment of cancer.
  • the agent typically exerts its effect by specifically inhibiting a protein, receptor or other molecular target which is involved in the process of angiogenesis.
  • An anti-angiogenic agent may also be referred to as an angiogenesis inhibitor or angiostatic agent.
  • the anti-angiogenic agent is a VEGF antagonist or inhibitor.
  • the agent is a biotherapeutic agent, for instance an immunoglobulin or fragment thereof, or a soluble receptor, e.g. a ligand trap such as Ziv-aflibercept (Zaltrap, VEGF Trap).
  • the agent is an antibody, especially a chimeric, humanized or fully human antibody.
  • the anti-angiogenic agent is bevacizumab (trade name Avastin ® , Genentech/Roche).
  • the anti-angiogenic agent may be a small molecule VEGF antagonist or inhibitor, for example a tyrosine kinase inhibitor or a VEGF receptor inhibitor.
  • Suitable anti-angiogenic agents include sorafenib (Nexavar, Bay 43-9006; 4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]- carbamoylamino]phenoxy]-N-methyl-pyridine-2-carboxamide), lenvatinib (4 ⁇ [3-chloro-4- (cyclopropylcarbamoylamino)phenoxy]-7-methoxy-quinoline-6-carboxamide), pazopanib (5-[[4-[(2,3- Dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzolsulfonamide), regorafenib (4-[4-( ⁇ [4-Chloro-3-
  • the present invention may be used to predict responsiveness to an anti-angiogenic therapy in the treatment of various cancers.
  • the cancer may be breast cancer, liver cancer, ovarian cancer, gastric cancer, bladder cancer, colon cancer, prostate cancer, lung cancer, nasopharyngeal carcinoma, cervical carcinoma, skin cancer, brain cancer including neuroblastoma and glioma, solid tumours, hematologic malignancies including leukemia and lymphoma, or head and neck cancer including squamous cell carcinoma of the lip, mouth, nasal cavity, pharynx, larynx, thyroid, paranasal sinuses, salivary glands or cervical lymph nodes of the neck.
  • ovarian cancer is particularly preferred for use in the present method.
  • the cancer may be colorectal cancer, breast cancer or renal cancer. Determining biomarker levels
  • biomarker levels are determined in a sample from the subject.
  • the amount of a particular biomarker in the sample may be measured by any suitable method.
  • methods for detecting protein biomarkers may include the use of an antibody, capture molecule, receptor, or fragment thereof which selectively binds to the protein.
  • Antibodies which bind to the biomarkers described herein are known or may be produced by methods known in the art, including immunization of an animal and collection of serum (to produce polyclonal antibodies) or spleen cells (to produce hybridomas by fusion with immortalised cell lines leading to monoclonal antibodies).
  • Detection molecules such as antibodies may optionally be bound to a solid support such as, for example, a plastic surface or beads or in an array.
  • Suitable test formats for detecting protein levels include, but are not limited to, an immunoassay such as an enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blotting and immunoprecipitation.
  • the level of the biomarker protein may be determined by mass spectroscopy.
  • Mass spectroscopy allows detection and quantification of an analyte by virtue of its molecular weight.
  • Any suitable ionization method in the field of mass spectroscopy known in the art can be employed, including but not limited to electron impact (El), chemical ionization (CI), field ionization (FDI), electrospray ionization (ESI), laser desorption ionization (LDI), matrix assisted laser desorption ionization (MALDI) and surface enhanced laser desorption ionization (SELDI).
  • Any suitable mass spectrometry detection method may be employed, for example quadrapole mass spectroscopy (QMS), fourier transform mass spectroscopy (FT-MS) and time-of-flight mass spectroscopy (TOF-MS).
  • sample used in the present method may be derived from various types of tissue.
  • the sample is derived from blood.
  • Suitable sample types include whole blood or any fractions of blood, including fractions which are cell free.
  • the sample comprises blood plasma or blood serum. Suitable methods for obtaining and fractionating blood samples are well known to those skilled in the art.
  • ocl-acid glycoprotein (AGP) AGP
  • Alpha-l-acid glycoprotein is a 41-kDa glycoprotein which functions as one of the major acute phase proteins in humans, rats, mice and other species (see e.g. Fournier et al., Biochim Biophys Acta. 2000; 1482(1-2):157-71). AGP is also known as orosomucoid (ORM). AGP is used herein to refer to both AGP-1 and AGP-2 (ORM-1 and ORM-2).
  • the amino acid sequence of human AGP-1 is available from, for example, database accession nos. GenBank AAB33887.1, RefSeq NP_000598.2 and UniProt P02763.
  • the amino acid sequence of human AGP-2 is available from, for example, database accession nos. GenBank AAH56239.1, RefSeq NP_000599.1 and UniProt P19652.
  • FLT4 Fms-like tyrosine kinase-4
  • FLT4 is a tyrosine kinase receptor for vascular endothelial growth factors C and D, and is also known as VEGF receptor 3 (VEGFR-3). FLT4 plays an essential role in adult lymphangiogenesis and regulates angiogenic sprouting (see e.g. Karkkainen et al., Oncogene 2000; 19(49):5598-605).
  • the amino acid sequence of human FLT4 is available from database accession nos. GenBank AAA85215.1, RefSeq NP_002011 and UniProt P35916.
  • Mesothelin is a 40kDa protein which is found on the mesothelial cells lining the pleura, peritoneum and pericardium in normal tissue and which may be overexpressed in cancer (see e.g. Hassan et al., Eur J Cancer. 2008; 44(1): 46-53).
  • the amino acid sequence of human mesothelin is available from database accession nos. GenBank AAH09272.1, RefSeq NP_001170826 and UniProt Q13421.
  • CA-125 is used herein to mean a member of the mucin family glycoproteins which may variously referred to as cancer antigen 125, carbohydrate antigen 125 or MUC16 (see Yin et al., Int J Cancer. 2002;98(5):737-40). CA-125 is commonly overexpressed in ovarian cancer patients.
  • the amino acid sequence of human CA-125 is available from database accession nos. RefSeq NP_078966.2 and UniProt Q8WXI7.
  • levels of at least two of the four biomarkers mentioned above are determined. It is demonstrated herein that whilst individual biomarkers may not be predictive of responsiveness to anti-angiogenic therapy per se, a biomarker signature comprising two or more biomarkers can be used to successfully discriminate responders from non-responders.
  • the method comprises determining a level of CA-125 and at least one further biomarker selected from the group consisting of AGP, FLT-4 and mesothelin. For instance, the method may comprise determining levels of at least (i) CA-125 and AGP; (ii) CA-125 and FLT-4; or (iii) CA-125 and mesothelin. In an alternative embodiment, the method may comprise determining a level of AGP and at least one further biomarker selected from the group consisting of CA-125, FLT-4 and mesothelin. For instance, the method may comprise determining levels of at least (i) AGP and mesothelin; or (ii) AGP and FLT-4.
  • the method may comprise determining a level of FLT-4 and at least one further biomarker selected from the group consisting of CA-125, AGP and mesothelin.
  • the method may comprise determining levels of at least FLT-4 and mesothelin.
  • the method may comprise determining a level of mesothelin and at least one further biomarker selected from the group consisting of CA-125, FLT-4 and AGP.
  • the method comprises determining at least 3 (i.e. 3 or 4) biomarkers from those mentioned above.
  • the method may comprise determining levels of (i) AGP, FLT-4 and mesothelin; (ii) AGP, FLT-4 and CA-125; (iii) AGP, mesothelin and CA-125; (iv) FLT-4, mesothelin and CA-125; or (v) AGP, FLT-4, mesothelin and CA-125.
  • two, three or four of the biomarkers mentioned above may be determined together with additional biomarkers which are considered to be indicative of responsiveness to anti- angiogenic therapy.
  • Additional biomarkers may, for example, be selected from VEGF-A, delta-like iigand-4, VEGF-C, neuropilin, VEGFRl and/or VEGFR2 single nucleotide polymorphisms, intercellular adhesion molecule (ICAM) and plasma placental growth factor.
  • IAM intercellular adhesion molecule
  • the levels of the biomarkers discussed above are compared to reference values.
  • a comparison is made with a predetermined or control value for that biomarker which provides an indication of responsiveness to therapy. For instance, whether the level of the biomarker in the sample is above or below the reference value may be used as an indicator of whether the subject is considered to be negative or positive for that particular biomarker. Responsiveness to anti-angiogenic therapy may then be determined by assessing how many of the determined biomarkers show a positive profile in the sample.
  • the reference value represents a cut-off or borderline value for a particular biomarker which separates subjects showing a tendency towards responsiveness from those showing a tendency towards non-responsiveness.
  • levels of individual biomarkers may not be capable of discriminating between responders and non- responders by themselves, subjects can be stratified into groups according to levels of each biomarker which is predictive when the results for two or more biomarkers are combined. Biomarker levels to either side of the cut-off value are therefore typically differentially associated with responsiveness to the anti-angiogenic therapy, even if this difference is only significant when the results are combined with those of a further biomarker having similar properties.
  • an increased level of al-acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4) and/or mesothelin in the sample from the subject, compared to its corresponding predetermined reference value is indicative of a positive result in the assessment of responsiveness to therapy.
  • AGP al-acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin in the sample from the subject is indicative of a positive result in the assessment of responsiveness to therapy.
  • an increased level of a particular biomarker (from those defined above) compared to the reference value may be indicative of responsiveness to the anti-angiogenic agent when combined with an increased level of a different biomarker (from those defined above).
  • increased levels of at least two or all three of al-acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4) and mesothelin is indicative of responsiveness to anti-angiogenic therapy.
  • AGP al-acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin is indicative of responsiveness to anti-angiogenic therapy.
  • a decreased level of cancer antigen 125 (CA-125) in the sample compared to the predetermined reference value is indicative of increased responsiveness to the anti-angiogenic agent.
  • the level of CA-125 in the sample may be compared to those of the other three biomarkers mentioned above in order to determine responsiveness to therapy. For instance, an increased level of ⁇ -acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4) and/or mesothelin compared to their predetermined reference values may be indicative of responsiveness to the anti-angiogenic agent when combined with a decreased level of CA-125 compared to its predetermined reference value.
  • AGP ⁇ -acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin compared to their predetermined reference values
  • the subject is predicted to be responsive to the anti-angiogenic agent if at least two of the following conditions (i) to (iv) apply to the subject:
  • the level of cancer antigen 125 (CA-125) in the sample is decreased compared to the predetermined reference value.
  • each of the four biomarkers may be determined and a subject scored as positive for that biomarker if (i) in the case of ⁇ -acid glycoprotein (AGP), fms-like tyrosine kinase-4 (FLT4) or mesothelin, the level of the biomarker in the sample is above the reference value; and (i) in the case of CA-125, the level of the biomarker in the sample is below the reference value.
  • AGP ⁇ -acid glycoprotein
  • FLT4 fms-like tyrosine kinase-4
  • mesothelin mesothelin
  • This provides a predictive index score for each subject on a scale from 0 to 4 (i.e. the subject is either positive for 0, 1, 2, 3 or 4 biomarkers).
  • a score of 3 or 4 may be taken as indicative of responsiveness to anti-angiogenic therapy, whereas a score of 0, 1 or 2 may be considered to be indicative of non-responsiveness of the subject.
  • the reference value for biomarker levels in serum may be (i) 1.0 to 1.6 g/l (e.g. 1.29 g/l) AGP; (ii) 30.0 to 60.0 ng/ml (e.g. 45.5 ng/ml) FLT-4; (iii) 0.5 to 1.5 nM (e.g. 0.96 nM) mesothelin; and/or (iv) 250 to 350 ku/l (e.g. 295 ku/l) CA-125.
  • the reference value is a particular value with one of the defined ranges, i.e.
  • the reference value is a specific numerical value rather than a range of values.
  • Alternative reference values may be selected according to the nature of the cancer, patient population, therapeutic strategy and so on. Treating cancer
  • the method described above may be used in order to select a treatment protocol for an individual subject. For instance, based on whether the subject is predicted to be responsive thereto, an anti-angiogenic therapy may be provided or an alternative treatment strategy employed.
  • the subject is treated with an anti-angiogenic agent (e.g. bevacizumab) if the method predicts that the subject will be responsive thereto.
  • the subject is typically treated with chemotherapy for cancer whether or not the anti-angiogenic agent is predicted to be effective. Thus subjects who are predicted to be responders are typically treated with a combination of the anti- angiogenic agent and chemotherapy, whereas predicted non-responders are treated with chemotherapy alone.
  • Chemotherapy typically refers to treatment with drugs or chemical compounds that target cancer cells. Chemotherapy may involve administration of a chemotherapeutic compound, which may have a cytotoxic or cytostatic effect, or which may induce a cyto-protective autophagy response in the cell.
  • the chemotherapeutic agent may be an agent that induces apoptosis, such as p53-dependent apoptosis, or that induces cell cycle arrest, including p53-dependent cell cycle arrest, in a cell that is abnormally proliferating or cancerous.
  • Commonly used chemotherapeutic agents include DNA damaging agents and genotoxic agents that can activate p53-dependent apoptosis or p53- dependent cell cycle arrest in a proliferating cell.
  • Suitable chemotherapeutic agents include an anthracycline, an alkylating agent, an alkyl sulfonate, an aziridine, an ethylenimine, a methylmelamine, a nitrogen mustard, a nitrosourea, an antimetabolite, a folic acid analogue, a purine analogue, a pyrimidine analogue, a podophyllotoxin, or a platinum-containing agent.
  • the chemotherapy may comprise administration of tamoxifen or a related taxane (e.g.
  • paclitaxel or docetaxel gemcitabine
  • carboplatin oxaliplatin
  • cisplatin erlotinib
  • adriamycin 5-fluoro uracil
  • etoposide camptothecin
  • camptothecin topotecan
  • liposomal doxorubicin or a derivative or analog thereof.
  • the chemotherapy comprises administration of carboplatin and/or paclitaxel.
  • chemotherapeutic agents include gemcitabine, oxaliplatin, cisplatin, topotecan, liposomal doxorubicin, docetaxel and erlotinib.
  • the subject may be treated with the anti-angiogenic agent (e.g. bevacizumab) in combination with radiotherapy.
  • the anti-angiogenic agent e.g. bevacizumab
  • Protocols for performing radiotherapy in e.g. ovarian cancer are well known to a skilled person, and may thus be combined with administration of an anti-angiogenic agent such as bevacizumab in subjects who are likely to be responsive thereto.
  • the anti-angiogenic agent may be formulated and administered to a subject in any suitable composition, optionally in combination with a chemotherapeutic agent for the treatment of cancer.
  • Administration of the anti-angiogenic agent in combination with a chemotherapeutic agent typically means that the administration of the anti-angiogenic agent occurs in a time period during which the subject is undergoing chemotherapy, for example simultaneously with, overlapping with, or sequentially prior to or following the administration of the chemotherapy.
  • the administration of the anti-angiogenic agent and the chemotherapy may each be achieved in one or more discrete treatments or may be performed continuously for a given time period required in order to achieve the desired result.
  • an effective amount of the anti-angiogenic agent is administered to the subject.
  • the term “effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to treat the specific cancer.
  • the anti-angiogenic agent may be administered to a subject using a variety of techniques.
  • the agent may be administered systemically, which includes by injection including intramuscularly or intravenously, orally, sublingually, transdermal ⁇ , subcutaneously, internasally.
  • the agent may be administered directly at a site at which the cancer is located. Delivery to the site includes topical administration, injection to the site, or surgical implantation, for example at a site of a tumour.
  • the anti-angiogenic agent is infused intravenously.
  • the concentration and amount of the anti-angiogenic agent to be administered will typically vary, depending on the cancer, the type of cell associated with the cancer, the type of agent that is administered, the mode of administration, and the age and health of the subject.
  • the anti-angiogenic agent may be formulated in a pharmaceutical composition together with a pharmaceutically acceptable diluent.
  • the compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives and various compatible carriers.
  • the anti-angiogenic agent may be formulated in a physiological buffer solution.
  • the proportion and identity of the pharmaceutically acceptable diluent may be determined by the chosen route of administration, compatibility with live cells, and standard pharmaceutical practice. Generally, the pharmaceutical composition will be formulated with components that will not - - significantly impair the biological properties of the agent. Suitable diluents are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • kits in further embodiments, provides a kit suitable for performing the method as described above.
  • the kit may comprise reagents suitable for detecting the biomarkers described above, e.g. at least 2, 3 or all 4 of the biomarkers.
  • the reagents may comprise antibodies which bind specifically to the biomarkers.
  • the kit may comprise two, three or four different antibodies, each of which binds to a different biomarker selected from those defined above.
  • kits may optionally further comprise one or more additional components, particularly reagents suitable for performing an ELISA assay using antibodies which bind to the biomarkers.
  • the kits may comprise capture and detection antibodies for each biomarker, secondary antibodies, detection reagents, solid phases (e.g.reaction plates or beads), standards (e.g. known concentrations of each biomarker in the form of recombinant proteins) as well as buffers suitable for performing any of step of an ELISA method.
  • the kits may further comprise vials, containers and other packaging materials for storing the above reagents, as well as instructions for performing a method as defined herein.
  • Serum samples from 10 patients within the bevacizumab arm of the ICON7 trial were selected for biomarker discovery, grouped as 5 responders (CR/PR) and 5 non-responders (SD/PD), with response defined by RECIST and/or CA-125 after 6 cycles of treatment. Selection on this basis was used as PFS data was not available at that time.
  • the patients in the two groups all had grade 3 serous tumours and were matched as closely as possible by age, FIGO disease stage and whether optimally or sub- optimally debulked at surgery (Table 4).
  • Paired serum samples at time-point 1 (baseline, prior to treatment with bevacizumab and chemotherapy) and timepoint 4 (pre-cycle 2) from each of the selected patients were subjected to proteomic analysis by label-free MS and candidate biomarkers of response selected.
  • 200 ⁇ of serum was filtered through 0.22 ⁇ Spin-X filters (Corning) by centrifugation for 1 minute at 16,000g and 150 ⁇ of filtered serum was depleted of the 14 most-abundant proteins using a Multiple Affinity Removal (MARS) human 14 column and depletion checked as previously described (7). Two runs were performed for each serum sample.
  • MAM Multiple Affinity Removal
  • Peptides triplicate injections each of 2 ⁇ g were separated by online reversed-phase capillary liquid chromatography (LC) and analyzed by electrospray tandem mass spectrometry (MS) using a Thermo Orbitrap Velos (9). Data were searched against an International Protein Index (IPI 3.80) human protein sequence database with MaxQuant 1.1.1.36 software (10) and the Andromeda search engine (11). The initial maximal mass tolerance for MS scan was set to 10 ppm, the fragment mass tolerance for MS/MS was set to 0.5 Th. The maximum protein and peptide false discovery rates were set to 0.01. Label-free quantitation was performed with MaxQuant.
  • IPI 3.80 International Protein Index
  • Results were subjected to initial exploratory data analysis using principal component analysis and hierarchical clustering considering the whole profile together to identify gross patterns and identify potential outliers. Subsequently each protein was examined separately using non-parametric tests and the q-value method to estimate the false discovery rate. Mann-Whitney tests were used to identify significant differences between treatment arms at time-points 1 and 4 and Wilcoxon signed- rank tests were used to identify differences in protein abundance between these time-points.
  • validation cohort I The mass spectrometry results from the discovery analysis were confirmed by immunoassay. Validation and exploration of initial findings was undertaken using a cohort of 92 patients (627 longitudinal samples (Table 1): validation cohort I), with further validation in an additional 115 patients (baseline samples only: validation cohort II). Samples in validation cohort I were selected to ensure similar numbers in each of the patient treatment and outcome groups described (limited by available assessable patients in the non-responder groups). Validation cohort II consisted of all remaining baseline samples from the biobank. Validation cohorts included patients from both arms of the trial (to enable markers differing specifically in response to bevacizumab and not just chemotherapy to be distinguished), were independent of each other and the discovery set and were representative of the trial population.
  • FLT4 (VEGFR3) ELISA A direct sandwich ELISA was developed for the quantification of soluble FLT4 (VEGFR3) using the human sVEGFR3 DuoSet (R&D Systems).
  • the coating antibody was used at 2 ⁇ g/mL and the biotinylated detection antibody at 0.25 ⁇ /mL, with detection using streptavidin-HRP (1:200; R&D systems) and tetramethylbenzadine (Sigma-Aldrich).
  • streptavidin-HRP (1:200; R&D systems
  • tetramethylbenzadine Sigma-Aldrich
  • the assay was standardized using a His-tagged recombinant human FLT4 protein (Sino Biological) with a standard curve range of 0.625-12.5 ng/mL.
  • the assay was optimized and fully validated with assessment of parallelism, recovery, cross reactivity, interference, hook effect, intra- and inter-assay precision and lower limit of quantification,
  • a sandwich ELISA kit was initially purchased for the quantification of soluble FLT4 (DuoSet, R&D Systems, Minneapolis, MN). This assay failed to validate when tested as serum FLT4 did not serially dilute in parallel with the assay standard. Four samples tested for parallelism gave results ranging 36.8-38.5% (acceptable limit is ⁇ 15%). Further investigation showed that this may be due to the tagged FLT4 protein used as a standard initially and when the original DuoSet standard was replaced - - with a polyhistidine-tagged recombinant protein (Sino-Biological, Beijing), parallelism to within acceptable limits was seen.
  • the FLT4 ELISA assay was validated using the monoclonal antibody pair from the DuoSet and the protein from Sino Biological.
  • the antibodies were initially tested in a checkerboard analysis to determine the ideal working concentrations, testing the capture antibody at 2, 4 and 6 ⁇ g/mL diluted in PBS and bound to a Nunc Maxisorp plate (Nalge Nunc International; Rochester, NY).
  • the detection antibody was tested at 0.25, 0.5 and 1 / ⁇ diluted in PBS-T (PBS, 0.1% v/v Tween 20).
  • PBS-T PBS, 0.1% v/v Tween 20
  • Several dilutions of recombinant protein (0, 0.313, 5.0 and 20 ng/mL) diluted in PBS-T + 1% w/v BSA were used to analyse the antibody performance.
  • Streptavidin HRP R&D systems
  • TMB tetramethylbenzadine
  • IN sulphuric acid Sigma-Aldrich
  • Antibody concentrations were selected as 2 ⁇ g/mL in PBS and 0.25 ⁇ g/mL in PBS-T for the capture and detection respectively.
  • the standard curve range was determined by running serial dilutions of recombinant protein from 30 ng/mL to 0.078 ng/mL and refined to a range of 12.5 ng/mL to 0.625 ng/mL.
  • multiple dilutions of serum samples with expected high and low FLT-4 concentrations were tested to determine a suitable sample dilution factor of 1 in 10.
  • the validation of the FLT4 ELISA was performed using serum samples from patients with diagnosed ovarian cancer.
  • the validation included: parallelism assessment; recovery analysis; intra- and inter- assay precision; lower limit of quantification determination; interference analysis; the assessment of hook effect and dilution linearity.
  • Parallelism assessment was conducted using three serum samples serially diluted from the initial dilution factor (1 in 10) three times to a final dilution of 1 in 80. Parallelism was deemed acceptable - - when the back calculated concentrations of each dilution gave a between dilution coefficient of variance of ⁇ 15%. All samples diluted parallel with results between 2.6 - 12.6%.
  • Recovery testing was performed using three serum samples spiked with low and high concentrations (final assay concentrations of 1.4 ng/mL and 6.7 ng/mL respectively) of recombinant FLT4 diluted in PBS-T + 0.1% w/v HSA. Spiked samples were compared to samples spiked to 10% of the total volume with recombinant protein diluent (PBS-T + 0.1% w/v HSA) and recombinant protein spiked into sample diluent to obtain the 'true' spike concentration. Recovery was found to be acceptable when the spiked sample adjusted for the endogenous FLT4 concentration was within 20% of the 'true' spike concentration. All samples recovered to between 81.5 - 104.9%.
  • Intra- and inter-assay precision were assessed using QC samples made from both ovarian cancer and healthy patient serum samples. Intra- and inter-assay precision, assessed on both QC samples run in duplicate using five independent dilutions on six plates, were found to be 4.2% and 3.6% (intra-) and 11.2% and 10.0% (inter-) for low and high QC samples respectively.
  • LLoQ Lower limit of quantification
  • the LLoQ of the assay was given as the concentration where the CV between the six measurements was ⁇ 20% and the mean concentration over the five measurements was 80-120% of the expected concentration, which in this case was 0.625 ng/mL.
  • the first part of interference was assessed by looking at common endogenous serum interferents. These included complement, rheumatoid factor, bilirubin, triglycerides and haemoglobin.
  • Haemolysis interference was tested by spiking pooled haemolysate (produced by washing EDTA plasma derived erythrocytes in PBS then lysing in MilliQ water) into a pool of serum samples to a haemoglobin concentration of 5 mg/mL. The sample was compared to the same pool of serum spiked with MilliQ water to the same ratio as the haemolysate spike. Interference was detected if the two samples differed by >20%. All other interferents were analysed by using serum samples with high concentrations of the relevant interferent spiked at 10% of the total sample volume to a final concentration of 5.4 ng/mL of recombinant FLT4. Samples were analysed as per recovery testing and interference was detected if the spiked sample adjusted for the endogenous FLT4 concentration was >20% of the true spike concentration. None of the interferents tested interfered with the assay performance. - -
  • Hook analysis was performed by spiking recombinant protein into serum samples to a final concentration of 5 ⁇ g/mL. Samples were then diluted 1 in 10 as per the assay protocol, making the spike concentration 500 ng/mL (40 times the top standard). Dilution linearity was assessed when samples were then diluted 1 in 100 to bring the sample back into the assay range (5 ng/mL). This test was mimicked in buffer as a comparison. It was confirmed that there was no hook effect, as spiked samples were not detected within the assay range. Samples diluted back into the range to the expected concentration (determined by the buffer test), it was therefore accepted that samples dilute linearly.
  • SM Ps Soluble mesothelin-related peptides
  • ESOMARK * assay Flujirebio Diagnostics
  • Serum AGP and CA-125 concentrations were measured using routine clinical assays by the Department of Clinical Biochemistry and Immunology (Leeds General Infirmary, UK). Serum AGP was measured on a Behring Nephelometric Analyser (BNA II, Siemens) and CA-125 levels were measured using the Siemens ADVIA Centaur CA-125 II assay.
  • BNA II Behring Nephelometric Analyser
  • the normal ranges for these assays are: AGP age ⁇ 50 female 0.4 - 1.0 g/L, male 0.6 - 1.2 g/L and age > 50 both sexes 0.8 - 2.0 g/L; CA-125 ⁇ 35 kU/L.
  • Results were examined for predictive utility, either on the basis of baseline concentrations or patterns of longitudinal change. All analyses were performed according to REMARK criteria (12). Associations between the 4 biomarkers under study were visualised using a correlogram based on simple linear regression and Spearman's rank correlation coefficient. Associations with demographic and clinical characteristics of the validation cohort at baseline were investigated using Fisher's exact test for categorical variables and Spearman's rank correlation coefficient for continuous variables. Corrections for multiple testing were not applied due to the explorative nature of the study.
  • Markers were considered initially as continuous variates and then as binary factors using cut-points derived by maximising Harrell's C-index (for visualisation of effects). The predictive potential of markers was assessed using interaction terms for treatment arm and biomarker concentration in Cox PH models.
  • An additive marker index with scale 0-4 was calculated from the individual markers.
  • the optimum cut-point in the index was identified using Cox PH models (by examining the likelihood ratio test on the interaction term for treatment arm and each level of the index), with indices below cut-points considered as signature-negative and those above as signature-positive.
  • PH assumptions were tested for each model using tests based on Schoenfeld residuals (14). All statistical tests were two-sided and all analyses were undertaken in the environment for statistical computing (15).
  • the markers showed no strong positive or negative associations (although correlations were significantly different from zero, all p ⁇ 0.01 in validation cohort I, Figure 6). Similar results were observed in validation cohort II and the combined validation cohort.
  • Concentrations of AGP also tended to reduce throughout the period of treatment, particularly between time-points 1-6, that is during chemotherapy (Figure 7a), with patients at high risk of progression showing the greatest reductions (Figure 7b).
  • the 4 groups were compared with available clinical characteristics which could be related to prognosis.
  • FIGO stage histology, ECOG performance status, surgical outcome (optimal or sub-optimal debulking) and those patients classified clinically to be at high risk of progression, there was no significant difference in the composition of the groups which could explain the better prognosis in the signature-negative, standard arm (all p>0.05, Fisher's exact test).
  • comparing age at randomization showed no significant difference between the four groups (p>0.05, Kruskal-Wallis test).
  • the final Cox PH model for the predictive index was internally validated using bootstrap resamples to estimate the optimism in the model (16).
  • the final model had an estimated Harrell's concordance index (C) of 0.62 and the optimism (O) was estimated to be 0.05 from 1000 resamples. As the estimated C-0 was >0.5, this implies that the final model has predictive ability.
  • biomarker index was re-calculated using the 115 time point 1 samples in validation cohort II with the same methods and cut-points as outlined above.
  • the biomarker index was recalculated using time point 1 samples from the 207 patients from validation cohorts I and II combined, using the same methods and cut-points as described above. - -
  • Bevacizumab has demonstrated efficacy in a number of different solid tumours (17-20), although it clearly does not benefit all patients and being able to identify the subset of patients who would derive most benefit would allow more selective appropriate use, hence improving bevacizumab efficacy and cost-effectiveness.
  • this exploratory translational study focussed on identification of relevant serum biomarkers in ICOIM7 patients, we identified FLT4, AGP and mesothelin using proteomic analysis, which we then sought to validate using immunoassays on a larger cohort of patients. Although baseline values of all candidate markers appeared to have prognostic value, unfortunately none displayed adequate characteristics to be predictive of benefit from the addition of bevacizumab over standard chemotherapy alone.
  • the candidate biomarkers were selected due to differential changes in protein concentrations seen between bevacizumab responders and non-responders by LC-MS/MS analysis.
  • the identification of a biomarker index with potential clinical utility demonstrates the validity of the approach.
  • Mesothelin is synthesised as a precursor that is cleaved to form soluble megakaryocyte potentiating factor (MPF; N-terminal fragment) and membrane bound mesothelin (C-terminal fragment).
  • MPF soluble megakaryocyte potentiating factor
  • C-terminal fragment membrane bound mesothelin
  • the immunoassay used detects forms including variant 1 most frequently found in ascitic fluid, due to shedding from ovarian cancer cells (22) and variant 3.
  • Mesothelin has been reported to be over-expressed in ovarian cancer and involved in tumour progression (23, 24) and associated with chemoresistance and a poorer overall prognosis (25).
  • Mesothelin has also been proposed as a potential biomarker for ovarian cancer identification (26, 27), and in mesothelioma as a putative biomarker to monitor response to treatment (28).
  • the association seen in this study between baseline mesothelin concentration and risk of ovarian cancer progression may relate to the increased disease burden in patients at higher risk of progression.
  • FLT4 (VEGFR3) is a tyrosine-protein kinase that acts as a cell-surface receptor for VEGF-C and VEGF- D, and plays a key role in angiogenesis by promoting proliferation, survival and migration of endothelial cells. It has a known pathological role in breast cancer angiogenesis (29). FLT4 signalling leads to increased VEGF-C, but also VEGF-A. In this study we saw a reduction of soluble FLT4 in patients treated with bevacizumab on the experimental arm, but this reduction was not seen in patients on the standard arm. This is likely to be due to the effect of the bevacizumab on the VEGF- related angiogenic pathway.
  • AGP is an acute phase protein which is found to be elevated in a number of different cancers (31, 32), although has not previously been reported of importance in ovarian cancer. It has however been reported to have pro-angiogenic properties and to support the pro-angiogenic effect of VEGF-A (33).
  • the results of the predictive biomarker index are derived from a randomised clinical trial, allowing a comparison between the standard (chemotherapy alone) and experimental (chemotherapy plus bevacizumab) arms.
  • Circulating protein and cellular biomarkers, single nucleotide polymorphisms (SNPs), expression arrays, and clinical correlates such as hypertension and dynamic contrast enhanced MRI parameters have been examined for clinical utility, in a variety of different diseases (reviewed in (34)) but no definitive biomarkers have yet been found.
  • Reasons for this are likely to include the complexity of angiogenesis and the limited understanding of the mechanism of action of bevacizumab, combined with the complications of combining bevacizumab with chemotherapy drugs which also have effects on relevant cellular pathways, limitations of study design (commonly - - being non-hypothesis driven, underpowered retrospective studies), and the use of archival primary tissue samples with potentially marked pre-analytical variation.
  • VEGF-A vascular endothelial growth factor-A
  • AVOREN renal cell carcinoma
  • Predictive biomarkers relating to bevacizumab in ovarian cancer are limited to smaller phase II studies and are only exploratory in nature (reviewed in (46)).
  • biomarkers relating to alternative angiogenic pathways found none to be of predictive value in a study of 106 patients with chemotherapy-resistant ovarian cancer treated with single agent bevacizumab (49).
  • Other putative biomarkers undergoing prospective testing include a VEGFR1 single nucleotide polymorphism (rs9582036), identified as predictive in renal cancer and pancreatic cancer from biomarker analyses relating to two phase III clinical trial involving bevacizumab (37).
  • bevacizumab is the first targeted therapy to be licensed for use in the first line treatment of OC, in combination with carboplatin and paclitaxel.
  • Current clinical practice is variable, with some clinicians using it in all patients with advanced OC, and others using it only in those clinically identified at high risk of progression.
  • biomarkers predictive of response to bevacizumab to enable avoidance of potential toxicities in those least likely to benefit and also to maximise the cost efficacy of this drug.
  • Our - - results demonstrate the utility of proteomic biomarker discovery in the initial identification of appropriate candidate biomarkers and the importance of sample collection alongside clinical trials to enable work such as this to be done.
  • a biomarker index has been identified which is predictive of benefit from bevacizumab, and which may be used to improve clinical outcomes by targeting bevacizumab therapy to responsive patients.
  • Table 1 Validation patient group baseline characteristics at randomization and outcomes.
  • Asian/Black/Other 7 1.24 (0.58, 5.21) 0.9508 50.02 (43.54, 59.46) 0.4407 1.29 (0.52,2.63) 0.8471 16 (5,970) 0.2803
  • Fallopian tube 3 1.17 (0.83, 3.98) 45.27 (35.85, 52.48) 1.27 (0.97,1.96) 104 (57,284)
  • Endometrioid 8 1.51 (0.96, 2.47) 57.41 (29.45, 103.40) 1.05 (0.54,2.74) 46 (7,181)
  • Table 3 Univariate and multivariable analysis of prognostic potential of biomarkers. Columns represent a univariate analysis in validation cohort followed by results when adjusting for risk in all patients and in each treatment arm separately. Univariate analysis of risk of progression in all patients and in each treatment arm separately is displayed in the fifth row of each cohort. HRs for biomarkers refer to increase in risk per unit increase in concentration.
  • PPC Primary peritoneal cancer
  • EOC Epithelial ovarian cancer
  • O optimal ( ⁇ 1 cm residual disease)
  • SO sub-optimal (>1 cm residual disease).
  • Table 5 Number of samples in each treatment arm at each time-point.

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Abstract

Selon un aspect, la présente invention concerne un procédé de prédiction de la réactivité d'un sujet à un agent anti-angiogénique pour le traitement du cancer, le procédé comprenant les étapes suivantes : (i) la détermination d'un niveau d'au moins deux biomarqueurs dans un échantillon prélevé du sujet, les biomarqueurs étant sélectionnés parmi le groupe constitué de la glycoprotéine alpha-1-acide (AGP), la tyrosine kinase analogue à fms 4(FLT4), la mésothéline et l'antigène CA-125 ; et (ii) la comparaison des niveaux des biomarqueurs dans l'échantillon avec des valeurs de référence prédéterminées ; le niveau des biomarqueurs dans l'échantillon comparés aux valeurs de référence prédéterminées indiquant la réactivité du sujet à l'agent anti-angiogénique pour le traitement du cancer.
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Publication number Priority date Publication date Assignee Title
WO2016183590A1 (fr) 2015-05-14 2016-11-17 Expression Pathology, Inc. Dosage par srm/mrm de la protéine mésothéline (msln)
JP2018519501A (ja) * 2015-05-14 2018-07-19 エクスプレッション、パソロジー、インコーポレイテッドＥｘｐｒｅｓｓｉｏｎ Ｐａｔｈｏｌｏｇｙ， Ｉｎｃ． メソテリン（ｍｓｌｎ）タンパク質のためのｓｒｍ／ｍｒｍアッセイ
EP3295180A4 (fr) * 2015-05-14 2018-10-17 Expression Pathology, Inc. Dosage par srm/mrm de la protéine mésothéline (msln)
AU2016262624B2 (en) * 2015-05-14 2019-11-07 Expression Pathology, Inc. SRM/MRM assay for the mesothelin (MSLN) protein

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