Method for predicting the outcome of a treatment with aflibercept of a patient suspected to suffer from a cancer

Abstract

The present invention concerns the use of interleukin-8 (IL-8) as a biomarker for predicting the outcome of the treatment with aflibercept, or ziv-aflibercept of a patient suspected to suffer from a cancer.

Description

<DP=1>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²1²METHOD FOR PREDICTING THE OUTCOME OF A TREATMENT WITH²AFLIBERCEPT OF A PATIENT SUSPECTED TO SUFFER FROM A CANCER²The present invention concerns the use of interleukin 8 (IL8) as a biomarker ²for predicting²the outcome of the treatment with aflibercept, or ziv-aflibercept, of a ²patient suspected to²suffer from cancer.²Aflibercept, or ziv-aflibercept, also referred to as VEGFR1R2-Fc.DELTA.C1²Flt1D2.F1k1D3.Fc.DELTA.C1 or AVE0005, is a homo dimer protein, with each dimer²comprising two identical monomers, each of which is a fusion protein ²comprising the²signal sequence of VEGFR1 fused to the D2 Ig domain of the VEGFR1 receptor, ²itself ²fused to the D3 Ig domain of the VEGFR2 receptor, in turn fused to the Fc ²domain of ²IgG1.²The protein chain is glycosylated, with N-acetyl-glucosamine, fucose, ²galactose, mannose²and sialic acids contributing to the carbohydrate structures. The N-linked ²oligosaccharides²consist of mainly bi-antennary structures with zero, one or two terminal ²sialic acids. The ²monomer has the amino acid sequence SEQ ID N 1.²The U.S. Food and Drug Administration (FDA) already approved aflibercept under ²the²trade name EYLEAO for the treatment of patients with neovascular (wet) age-²related²macular degeneration (AMD). In particular, EYLEAO is the trade name for ²aflibercept as ²generated, processed and formulated for intravitreal injection.²At the time of registration of aflibercept (zaltraP) for cancer indication, ²and In light of ²aflibercept's approved use in treating AMD, the FDA requested that a different ²name (ziv-²aflibercept) be given for the compound's use in the treatment of cancer. Thus, ²ziv-²aflibercept is the United States Adopted Name (USAN) accepted by FDA to ²designate a ²pharmaceutical composition comprising aflibercept as generated, processed and ²formulated for injection via intravenous infusion. Ziv-aflibercept has been ²approved by the ²FDA for sale under the tradename ZALTRAPO for the treatment of metastatic ²colorectal² cancer (mCRC).²The European Medicines Agency (EMA) approved zaltrap as well however did not ²request ²separate names for the compound. Thus, in the European Union the name ²"aflibercept" is ²used regardless of the indication.²<DP=2>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²2²ZALTRAPO and EYLEAO are obtained by slightly different processes. They both ²contain ²aflibercept or ziv-aflibercept, but the ratio of aggregates of aflibercept or ²ziv-aflibercept is ²slightly different in ZALTRAPO and EYLEAO.²ZALTRAPO approval was based on data obtained from the VELOUR trial ¨ a ²multicenter,²randomized, placebo-controlled phase III trial, which compared the efficacy of ²aflibercept²versus placebo in combination with the FOLFIRI regimen for patients with mCRC ²previously treated with an oxaliplatin containing regimen.²AFFIRM, an open-label, non-comparative, phase II study, was conducted to ²assess the ²combination of aflibercept and modified FOLFOX6 (mFOLFOX6) given as first-line²therapy in patients with mCRC. The primary endpoint was 12-month progression-²free²survival (PFS) whereas exploration of biomarkers was among the secondary ²objectives.²In an attempt to understand the key factors associated with aflibercept ²efficacy and safety, ²an investigation assessing biomarkers for aflibercept treatment in ²prospectively collected, ²tumor tissues and serially sampled plasma from patients participating in the ²AFFIRM² study was conducted.²Plasma proteins and genetic variants, representing either single nucleotide ²polymorphisms (SNPs) in angiogenicpathway genes or somatic mutations in key ²oncogenic drivers of mCRC, were analyzed to assess if they could predict ²response to ²aflibercept with respect to PFS. Subsequently, one also assessed whether any ²of these²markers correlated with anti-angiogenic drug-induced AEs, such as ²gastrointestinal²perforation, thrombosis, hypertension and proteinuria.²Despites the efficacy and the safety of the treatment of cancer by aflibercept ²it remains a ²goal to better identify patients who should benefit more from the treatment.²It has now been discovered that high IL8 levels at baseline correlated with ²shorter survival ²times, and patients with increasing levels of IL8 during treatment were more ²likely to ²progress. This suggests that patients with high IL8 levels, at baseline or ²during treatment, ²are at increased risk of disease progression during aflibercept therapy.²The role of 1L8-in tumor development and progression is suggested in the prior ²art²In a phase II trial hepatocellular carcinoma patients received bevacizumab ²(Boige V, ²MaIke D, Bourredjem A et al. Efficacy, safety, and biomarkers of single-agent ²bevacizumab therapy in patients with advanced hepatocellular carcinoma. ²Oncologist ²2012; 17: 1063-1072). Circulating endothelial cells (CECs) and plasma ²cytokines and² angiogenic factors (CAFs) were measured at baseline and throughout treatment.²<DP=3>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²3²This study showed that elevated baseline IL-8 (above 80 pg / ml) and IL-6 levels ²were ²correlated with both a shorter progression-free survival (PFS) interval and a ²shorter ²overall survival (OS) time.²However the authors cite another study with sorafenib wherein conversely, IL-8 ²was the² only serum cytokine not correlated with the PFS outcome.²In another phase II trial metastatic colorectal cancer (mCRC) patients ²received ²bevacizumab (Kopetz S, Hoff PM, Morris JS et al. Phase II trial of infusional ²fluorouracil, ²irinotecan, and bevacizumab for metastatic colorectal cancer: efficacy and ²circulating²angiogenic biomarkers associated with therapeutic resistance. J Olin Oncol ²2010; 28: 453-²459).²Levels of 37 CAFs were assessed at baseline, during treatment, and at the time ²of ²progressive disease (PD).²The authors conclude that elevated baseline IL8 above the median value of 3.7 ²pg / ml was²associated with shorter PFS times.²These results were obtained with bevacizumab which is an antibody. Aflibercept ²is ²not an antibody but a fusion protein and thus has a different mode of action.²It is not possible to predict the effect of a biomarker on the efficiency of ²cancer ²treatment by a given drug from results obtained with another drug.²A fortiori the threshold above or under which a patient is considered as a ²candidate²for treatment cannot be extrapolated between two different drugs.²The invention relates to the use of interleukin-8 (IL-8) as a biomarker for ²predicting ²the outcome of the treatment with aflibercept, or ziv-aflibercept of a patient ²suspected to ²suffer from a cancer.²In one aspect, the present invention provides a method of determining whether ²a²patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-²aflibercept ²therapy for the said cancer comprising the step of subjecting a patient's ²biological sample ²to at least one assay to measure at baseline the IL-8 level, wherein when the ²biological ²sample IL-8 level is low relative to a reference level of expression of IL-8, ²the patient is²identified as a candidate for therapy for cancer.²In another aspect, the present invention provides a method of determining ²whether a ²patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-²aflibercept ²therapy for the said cancer comprising the step of subjecting a patient's ²biological sample ²to at least one assay to measure at baseline the IL-8 level, wherein when the ²biological²<DP=4>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²4²sample IL-8 level is high relative to a reference level of expression of IL-8, ²the patient is ²identified as not being a candidate for therapy for cancer.²In an embodiment the reference level of expression of IL-8 is comprised ²between around ² and around 30 pg / ml.²5 In a further embodiment the reference level of expression of IL-8 is ²comprised between²around 15 and around 25 pg / ml or around 17 and around 21 pg / ml.²In a further embodiment the reference level of expression of IL-8 is around 18 ²pg / ml, ²around 19 pg / ml or around 20 pg / ml.²The invention relates also to a method for treating a patient with a cancer ²with aflibercept, ²10 or ziv-aflibercept, comprising administering a therapeutically effective ²amount of ²aflibercept, or ziv-aflibercept to the patient, wherein the IL-8 level in the ²patient's biological ²sample is low relative to a reference level of expression of IL-8.²The invention further relates to a method of optimizing therapeutic efficacy ²for treatment of ²a cancer, comprising the steps of:²a) administering aflibercept, or ziv-aflibercept to a patient suspected to ²suffer from a²cancer; and²b) determining the level of interleukin-8 (IL-8) in patient's biological ²sample,²wherein an increase of the interleukin-8 (IL-8) level indicates the need to ²decrease the ²amount of aflibercept, or ziv-aflibercept in subsequent administrations.²A further object of the invention is a method of managing the risk to allow a ²safe use of²aflibercept, or ziv-aflibercept in the treatment of a patient suspected to ²suffer from a ²cancer, said method comprising the following steps:²a) before beginning of the treatment with aflibercept, or ziv-aflibercept, ²determining the interleukin-8 (IL-8) level in a biological sample from the²patient;²b) along the treatment determining the interleukin-8 (IL-8) level in a ²biological sample from the patient²c) comparing the interleukin-8 (IL-8) level determined in step (b) with the ²level determined in step (a),²<DP=5>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²whereby a higher interleukin-8 (IL-8) level in the sample of step (b) compared ²to that of²level in the sample of step (a) indicates that the patient should be closely ²monitored.²In an embodiment of one of the methods described above the biological sample ²is chosen² 5 from the group consisting of blood, serum and plasma.²In an embodiment of one of the methods described above the cancer is a colon ²cancer, a ²colorectal cancer or a rectal cancer.²In a further embodiment of one of the the colorectal cancer is a metastatic ²colorectal ²cancer.²In another embodiment of the invention, the subject is treated with ²aflibercept and ²further undergoes a chemotherapeutic treatment with oxaliplatin, 5-²fluorouracil (5-FU) and ²folinic acid (i.e. the FOLFOX treatment), folinic acid, 5-fluorouracil and ²irinocetan (i.e. the ²FOLFIRI treatment), or 5-fluorouracil and folinic acid (i.e. the FUFOL or ²LV5FU2²treatment).²The chemotherapeutic treatment may combine at least 2, 3, 4, 5, 6, 7, 8, 9, 10 ²or at ²most 10, 9, 8, 7, 6, 5, 4, 3, 2 agents, such as e.g. a combination of ²oxaliplatin, 5-²fluorouracil (5-FU) and folinic acid (i.e. the FOLFOX treatment or the ²modified FOLFOX6 ²treatment as described in the example below), a combination of folinic acid, 5-²fluorouracil²and irinocetan (i.e. the FOLFIRI treatment), or a combination of 5-²fluorouracil and folinic²acid (i.e. the FUFOL or LV5FU2 treatment).²In this regard the application W02012146610 relates to a method of treatment ²of ²the mCRC by aflibercept, or ziv-aflibercept in combination with FOLFIRI. The ²content of ²this application is incorporated by reference.²In an embodiment of one of the methods described above therapeutically ²effective ²amounts of aflibercept, or ziv-aflibercept, oxaliplatin, 5-fluorouracil (5-FU) ²and folinic acid ²are administered to said patient.²In an embodiment of one of the methods described above therapeutically ²effective²amounts of aflibercept, or ziv-aflibercept, folinic acid, 5- fluorouracil (5-²FU) and irinocetan²are administered to said patient.²In a further embodiment of one of the methods described above folinic acid at ²a dosage²comprised between about 200 mg / m2 and about 600 mg / m2, 5-fluorouracil (5-FU) ²at a²dosage comprised between about 2000 mg / m2 and about 4000 mg / m2, irinocetan at ²a²dosage comprised between about 100 mg / m2 and about 300 mg / m2 and aflibercept ²at a²<DP=6>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²6²dosage comprised between about 1 mg / kg and about 10 mg / kg are administered to ²patient.²In a further embodiment of one of the methods described above folinic acid at ²a dosage of ²about 400 mg / m2, 5-fluorouracil (5-FU) at a dosage of about 2800 mg / m2, ²irinocetan at a² dosage of about 180 mg / m2 and aflibercept at a dosage of about 4 mg / kg are²administered to patient.²In a further embodiment of one of the methods described above folinic acid is²administered intravenously at a dosage of about 400 mg / m2, 5-fluorouracil (5-²FU) is ²administered intravenously at a dosage of about 2800 mg / m2, irinocetan is ²administered² intravenously at a dosage of about 180 mg / m2 and aflibercept is administered²intravenously at a dosage of about 4 mg / kg and wherein the combination is ²administered ²every two weeks.²In a further embodiment of one of the methods described above folinic acid, 5- ²fluorouracil ²(5-FU), irinocetan and aflibercept are administered intravenously every two ²weeks for a²period comprised between 9 and 18 weeks.²In a further embodiment of one of the methods described above folinic acid is ²administered intravenously immediately after aflibercept administration. It ²can be also ²administered intravenously immediately after aflibercept administration over a ²period of ²about 2 hours.²In a further embodiment of one of the methods described above irinocetan is ²administered²intravenously immediately after aflibercept administration. It can be also ²administered²intravenously immediately after aflibercept administration over a period of ²about 90 ²minutes.²In a further embodiment of one of the methods described above 5-fluorouracil ²(5- FU) is²administered immediately after aflibercept administration.²In a further embodiment of one of the methods described above a first quantity ²of 5-²fluorouracil (5-FU) is administered intravenously immediately after ²aflibercept ²administration and a second quantity of 5-FU is administered intravenously ²after the first ²quantity in continous infusion.²<DP=7>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²7²In a further embodiment of one of the methods described above about 400 mg / m2 ²of 5-²fluorouracil (5-FU) is administered intravenously over a period of 2 to 4 ²minutes after ²aflibercept administration and wherein 2400 mg / m2 of 5-FU is administered ²intravenously ²over around 46 hours after the administration of the 400 mg / m2 in continous ²infusion.²In an embodiment said patient has previously been treated with therapy based ²on²oxaliplatin or on bevacizumab.²In another embodiment said patient has failed with chemotherapy, radiotherapy ²or ²surgery.²The invention relates also to aflibercept, or ziv-aflibercept for treating a ²patient suspected²to suffer from cancer, wherein the IL-8 level in the patient's biological ²sample is lower than²between around 15 and around 50 pg / ml.²The invention further relates to a kit for predicting whether a patient ²suspected to suffer ²from cancer is a candidate for aflibercept, or ziv-aflibercept therapy, which ²kit comprises:²a) means for measuring the interleukin-8 (IL-8) level; and²b) Optionally, a label giving instructions for the use of said kit in ²predicting whether a²patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-²²aflibercept therapy.²Another aspect of the invention further relates to an article of manufacture ²comprising:² a) a packaging material;²b) means for measuring the interleukin-8 (IL-8) level; and²c) a label giving instructions for the use of said kit in predicting whether a ²a patient ²suspected to suffer from cancer is a candidate for aflibercept, or ziv-²aflibercept ²therapy.²The above methods and use of the invention may be, for instance, in vitro or ²ex ²vivo methods and use.²Means for measuring the expression level of IL8 protein are well-known in the ²art²and include immunoassay such as ELISA assay. The means for measuring IL8 ²protein ²include antibodies specifically binding to IL8. Such means can be labeled with ²detectable ²compound such as fluorophores or radioactive compounds. For example, the probe ²or the²<DP=8>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²8²antibody specifically binding to IL8 may be labeled with a detectable ²compound. ²Alternatively, when the kit comprises an antibody, the kit may further ²comprise a ²secondary antibody, labeled with a detectable compound, which binds to an ²unlabeled ²antibody specifically binding to IL8.² The means for measuring the expression level of IL8 may also include reagents²such as e.g. reaction and / or washing buffers. The means may be present, e.g., ²in vials or ²microtiter plates, or be attached to a solid support such as a microarray as ²can be the ²case for primers and probes.²Aflibercept, or ziv-aflibercept is provided in a formulation which is not ²prejudicial to²the patient to be treated.²In an embodiment aflibercept, or ziv-aflibercept is provided in a formulation ²with ²sucrose and polysorbate 20 (stabilisers), sodium chloride, citrate buffer, and ²sodium ²phosphate buffer, adjusted to final pH.²In another embodiment aflibercept, or ziv-aflibercept, is supplied in two drug²product presentations:²- a presentation at 100 mg aflibercept, or ziv-aflibercept / 4.0 mL (nominal ²concentration).²- a second presentation at 200 mg aflibercept, or ziv-aflibercept / 8.0 mL ²(nominal²concentration).² Both presentations are manufactured from the same bulk sterile solution at 25²mg / mL of aflibercept, or ziv-aflibercept.²Prior to infusion to the patient, the concentrate solution is diluted with ²0.9% sodium ²chloride solution or 5% dextrose.²The anti-cancer agents used in the above recited method or use are provided in ²a²pharmaceutically acceptable carrier, excipient or diluent which is not ²prejudicial to the²patient to be treated.²Pharmaceutically acceptable carriers and excipient that may be used in the ²compositions of this invention include, but are not limited to, ion ²exchangers, alumina, ²aluminium stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) ²such as d-a-²tocopherol polyethyleneglycol 1000 succinate, surfactants used in ²pharmaceutical dosage²forms such as Tweens or other similar polymeric delivery matrices, serum ²proteins, such ²as human serum albumin, buffer substances such as phosphates, glycine, sorbic ²acid, ²potassium sorbate, partial glyceride mixtures of saturated vegetable fatty ²acids, water, ²salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, ²potassium²hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium ²trisilicate,²polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium²<DP=9>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²9²carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-²block ²polymers, polyethylene glycol and wool fat.²As appreciated by skilled artisans, compositions are suitably formulated to be ²²compatible with the intended route of administration. Examples of suitable ²routes of²administration include parenteral route, including for instance intramuscular, ²²subcutaneous, intravenous, intraperitoneal or local intratumoral injections. ²The oral route ²can also be used, provided that the composition is in a form suitable for oral ²²administration, able to protect the active principle from the gastric and ²intestinal enzymes.²The terms "Therapy", "therapeutic", "treatment" and "treating" are used herein ²to²characterize a therapeutic method or process that is aimed at (1) slowing down ²or²stopping the progression, aggravation, or deterioration of the symptoms of the ²disease ²state or condition to which such term applies; (2) alleviating or bringing ²about ²ameliorations of the symptoms of the disease state or condition to which such ²term ²applies; and / or (3) reversing or curing the disease state or condition to ²which such term² applies.²As used herein, the terms "interleukin-8" and "IL-8" are used interchangeably ²and ²refer to all of the naturally-occurring isoforms, including alternative splice ²variants, allelic ²variants and include naturally occurring variants, SNPs (single nucleotide ²polymorphisms), ²and truncated or secreted forms of IL-8 protein.²In particular, the terms "interleukin 8" refers to the polypeptide comprising ²or²consisting of the amino acid sequence corresponding to the UniProtKB / Swiss-²Prot ²accession number P10145 (SEQ ID NO: 2) and / or²a) a polypeptide corresponding to the mature isoform of the polypeptide ²of (a) (i.e. obtained after cleavage of the signal peptide); and / or²b) an allelic variant of the polypeptide of (a) or (b); and / or²c) a splice variant of a polypeptide of (a), (b) or (c); and / or²d) a constitutively active mutant of a polypeptide of (a), (b), (c) or (d).²e) an isoform obtained by proteolytic processing of a polypeptide of (a), ²(b), (c), (d) or (e).²By "isoform of a polypeptide" is meant a polypeptide that has at least about ²50%,²55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, ²82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, ²97%, 98%, or 99% amino acid sequence identity to a full-length polypeptide ²reference ²sequence and has the same biological activity. In the context of the present ²application,²the percentage of identity is calculated using a global alignment (i.e. the ²two sequences²<DP=10>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²are compared over their entire length). Methods for comparing the identity of ²two or more ²sequences are well known in the art. The needle program, which uses the ²Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. ²Mol. ²Biol. 48:443-453) to find the optimum alignment (including gaps) of two ²sequences when²5 considering their entire length, may for example be used. The needle ²program is for²example available on the ebi.ac.uk World Wide Web site. The percentage of ²identity in ²accordance with the invention can be calculated using the EMBOSS::needle ²(global) ²program with a "Gap Open" parameter equal to 10.0, a "Gap Extend" parameter ²equal to ²0.5, and a Blosum62 matrix.²10 "Isoform" also refers to all post-translationally modified forms of IL8 ²protein. Post-²translationally modified isoforms may include acetylated, formylated, ²lipoylated, ²myristoylated, palm itoylated, alkylated, methylated, amidated, glycosylated, ²hyrdroxylated, ²nitrosylated, phosphorylated, sulphated, polysialylated and sialylated forms.²The "reference level of expression of IL-8" may be determined as a single ²value or²a range of values which is determined based on the expression level of IL-8 ²measured, for²instance, in a population of healthy subjects or in a population of subjects ²in need of an ²aflibercept therapy.²In an embodiment the reference level of expression of IL-8 is determined based ²on ²the expression level of IL-8 measured in a population of subjects in need of a ²aflibercept ² therapy.²Typically, the analysed population could be divided into percentiles based on ²the ²measured level of expression of IL-8. The reference level could be defined as ²the ²percentile that provides the best separation between patients suffering from a ²cancer on ²which the treatment with aflibercept is efficient and patients suffering from ²a cancer on²which the treatment with aflibercept is not efficient enough to cure it.²In the study reported in Example 1 below, the reference level of expression of ²IL-8 ²was 19 pg / ml (77th percentile).²However, the reference level of expression of IL-8 may vary i) according to ²the size ²of the studied population, and ii) depending on the method used for measuring ²the IL-8 ²expression.²The level of interleukin 8 protein may be, for instance, determined using ²immunological detection methods such as an ELISA assay. The methods involve an ²²antibody which binds to interleukin 8 protein, for example a monoclonal or ²polyclonal ²antibody, an antibody variant or fragments such as a single chain antibody, a ²diabody, a²minibody, a single chain Fv fragment (sc(Fv)), a Sc(Fv)2 antibody, a Fab ²fragment or a²F(ab')2 fragment, or a single domain antibody. Such antibodies are well known ²in the art²<DP=11>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²11²and are commercially available. They may also notably be obtained by ²immunization of ²animals (for example rabbits, rats or mice) with interleukin 8 protein. ²Antibodies may be ²used to determine protein expression in a range of immunological assays ²including ²competitive and non-competitive assay systems using techniques such as western²blotting, immunohistochemistry / immunofluorescence (i.e protein detection on ²fixed cells²or tissues), radioimmunoassay such as RIA (radio-linked immunoassay), ELISA ²(enzyme ²linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation ²assays, ²immunodiffusion assays, agglutination assays, complement-fixation assays, ²immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-²linked² immunoassay), chemiluminescence immunoassays, ECLIA (electrochemiluminescence²immunoassay) and protein A immunoassays. Such assays are routine and well ²known to ²the person skilled in the art (Ausubel et al (1994) Current Protocols in ²Molecular Biology, ²Vol. 1, John Wiley & Sons, Inc., New York).²Protein expression of interleukin 8 may also be determined by proteomic method²such as mass spectrometry assays (LC-MS or LC-MS / MS). Qualitative and ²quantitative ²mass spectrometric techniques are known and used in the art. To this aim, ²target peptides ²specific for marker proteins are selected and quantified based on calibration ²curves ²established with synthetic peptides labeled with stable isotopes. Enzymatic ²digests,²spiked with a defined amount of isotope labeled target peptides, are analyzed ²by liquid²chromatography coupled with mass spectrometry. The ratio between labeled and ²non-²labeled target peptides is measured to assess target peptide concentrations ²and therefore ²protein marker concentration.²The expression "circulating IL8" is intended to mean the IL8 proteins present ²in²blood, serum and plasma.²A "subject" or a "patient" may be a human or a non-human mammal, such as²monkeys, dogs, cats, guinea pigs, hamsters, rabbits, cows, horses, goats and ²sheep.²Figures 1 and 2 illustrate the relation between IL8 levels and the probability ²of²disease progression. Depicted is the probability of disease progression after ²12 months in ²relation to IL-8 plasma levels at baseline (Figure 1) and the difference ²between 1L8 ²plasma levels at baseline and the last measurement point before disease ²progression ²(Figure 2). Briefly, figure 1 shows that high IL8 levels correlate with ²increased probability²for disease progression and that this effect is slightly more pronounced in ²aflibercept²versus FOLFOX treated patients. On the other hand, figure 2 shows that the ²increase in²<DP=12>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²12²IL8 relative to baseline also corresponds to disease progression. Since ²increases in IL8 ²are plotted on a logarithmic scale, values between -3 and 0 correspond to an ²increase in ²IL8 < 1 pg / mL, whereas values between 0 and 3 represent increases >1 pg / mL. ²Data thus ²show that even small increases in IL8 relative to baseline already correspond ²to an² increased probability of disease progression in the aflibercept arm.²EXAMPLE: Effect of interleukin 8 on PFS in the AFFIRM Study ² Study ECF10668 (AFFIRM) ²EFC10668 was designed as a randomized, multinational, study comparing the ²adverse effects occurrence in patient with metastatic colorectal cancer (MCRC) ²treated ²with:²i) a modified FOLFOX6 (a combination of oxaliplatin, 5-fluorouracil (5-FU) and²folinic acid) given intravenously every 2 weeks as first-line treatment (arm ²A); or²ii) aflibercept at 4 mg / kg combined with a modified FOLFOX6 given ²intravenously ²every 2 weeks as first-line treatment; or²Schedule of administration² Patients were administered intravenously either with aflibercept immediately²followed by oxaliplatin, 5-fluorouracil (5-FU) and folinic acid (modified ²FOLFOX6 regimen) ²or modified FOLFOX6 alone , depending on arm to which they were assigned,²This treatment was repeated every 2 weeks until progression (or unacceptable ²toxicity, or consent withdrawal).² Dosage²The patients randomized in the aflibercept arm received 4mg / kg IV every 2 ²weeks.²The following were administered to patients in both treatment groups:²= Oxaliplatin (Eloxatinq² = Folinic acid (also known as leucovorin)²= 5-fluorouracil²Formulations of oxaliplatin, 5-fluorouracil, and folinic acid:²= Products used were those available in the hospital / clinic pharmacy²= Route of administration: IV²<DP=13>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²13²Dose: Oxaliplatin, folinic acid, and 5-fluorouracil were administered ²according to ²an mFOLFOX6 regimen, as follows:²= Oxaliplatin 85 mg / m2 as a 2-hour IV infusion on day 1²= Folinic acid 350 mg / m2 as a 2-hour IV infusion on day 1²= 5-fluorouracil ²400 mg / m2 as an IV bolus on day 1, and then 2400²mg / m2 as a 46-hour continuous IV infusion starting on day 1²In case of body surface area >2.0 m2, the actual doses of oxaliplatin and of 5-²FU ²were to be adjusted to a maximum BSA of 2.0 m2 for safety reasons. Dose ²reduction ²and / or treatment delay and / or treatment discontinuation were planned in case ²of severe²toxicity. The modified FOLFOX6 regimen was administered after administration ²of²aflibercept.²Duration of treatment:²Treatment for an individual patient was administered up until progression or ²until² unacceptable toxicity occurred or the patient withdrew consent.²Treatment duration was estimated to be approximately 12 months.²Demographics and baseline characteristics ²Table 1 below compares demographics and patient characteristics at baseline² between biomarkers evaluable and non-evaluable populations.²The "biomarkers evaluable population" is defined as the population of patients ²who ²provided a blood / tumor sample for biomarker assessment; while the ²"biomarkers non ²evaluable population" corresponds to patients who did not provide blood / ²tumor sample ²for biomarker assessment (e.g. patients who did not consent to biomarker ²study).²All characteristics are similar between populations, except for the region of ²origin²of the patients: Eastern Europe tends to be over-represented and other ²countries tend to ²be under-represented in the biomarkers evaluable population compared to the ²biomarker ²non evaluable population.²Table 1: Summary of patient demographics and patient characteristics at ²baseline²- Evaluable population²Biomarkers non evaluable Biomarkers evaluable²population population p-value²Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70) ²<DP=14>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²14²Biomarkers non evaluable Biomarkers evaluable²population population p-value²Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70)²Gender²[n(%)] 1.0000a²Number 57 49 60 70²33 36 43²Male 32(56.1%) (67.3%) (60.0%) (61.4%)²16 24 27²Female 25 (43.9%) (32.7%) (40.0%) (38.6%)²Age (Years) 0.2811b²Number 57 49 60 70²Median 66.0 62.0 62.0 62.5²Mean²(SD) 63.7 (10.0) 61.8 (9.5) 61.3 (9.4) 61.7 ²(8.7)²Min : Max 44 : 87 29 : 75 37 : 81 41: 79²Age class²[n(%)] 0.2421a²Number 57 49 60 70²28 38 42²<65 27 (47.4%) (57.1%) (63.3%) (60.0%)²65 but 20 20 25²<75 23 (40.4%) (40.8%) (33.3%) (35.7%)²75 7 (12.3%) 1 (2.0%) 2 (3.3%) 3 (4.3%)²Race [n(%)] 0.0640a²Number 57 49 60 70²Caucasian 37 49 60² / White 41(71.9%) (75.5%) (81.7%) (85.7%)²Black 0 0 0 1 (1.4%)²Asian / One 11 11 9²ntal 16 (28.1%) (22.4%) (18.3%) (12.9%)²Other 0 1 (2.0%) 0 0²Region 0.0029a²Number 57 49 60 70²Western 30 37 45²Europe 37(64.9%) (61.2%) (61.7%) (64.3%)²Eastern 12 12²Europe 3 (5.3%) 3 (6.1%) (20.0%) (17.1%)²Other 16 11 13²countries 17 (29.8%) (32.7%) (18.3%) (18.6%) ²<DP=15>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262² Biomarkers non evaluable Biomarkers evaluable²population population p-²value² Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70)²BSA (m2) ²0.1091b²Number 57 49 60 70²Median 1.8 1.8 1.8 1.8²Mean²(SD) 1.7 (0.2) 1.8 (0.2) 1.8 (0.2) 1.8 (0.2)²Min : Max 1 : 2 1 : 2 1 : 2 1 : 2²Weight (kg) ²0.0838b²Number 57 49 60 70²Median 67.6 70.0 73.2 71.4²Mean²(SD) 67.3 (14.1) 71.1 (16.6) 74.0 (17.0) 71.5 ²(15.6)²Min : Max 40 : 107 40 : 115 48 : 134 40 : 117²'comparing frequency distribution based on Fisher's exact test - 2-sided. ²busing ANOVA ²(type 3) with factors:BIOPOP, BIOPOP. Records with missing values for factors ²or ²response were excluded from statistical analyses.²5 Frequency distribution of covariates is compared between evaluable and ²non-evaluable²populations²Note: Western Europe = Germany, Italy, Spain, United Kingdom; Eastern Europe = ²²Russian Federation; Other countries = Australia, Korea² 10 Disease characteristics at baseline ²Disease characteristics at baseline were similar in the two populations (see ²Tables ²2 and 3 below).²Table 2: Summary of disease characteristics at initial diagnosis² Biomarkers non Biomarkers evaluable²evaluable population population p-²value² Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70)²Primary site²[n(%)] ²0.7045a²Number 57 49 60 70²Colon 27 (47.4%) 26 (53.1%) 31(51.7%) ² 33 (47.1%)²Recto sigmoid 9 (15.8%) 15 (30.6%) 16 (26.7%) ² 19 (27.1%)²Rectum 21(36.8%) 8 (16.3%) 13 (21.7%) 18 (25.7%) ²<DP=16>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²16²Biomarkers non Biomarkers evaluable²evaluable population population p-value²Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70)²Histology type ²[n(%)]²Number 57 49 60 70²Adenocarcino²ma 57 (100%) 49 (100%) 60 (100%) 70 (100%)²Staging at ²diagnosis²[n(%)] 0.2297a²Number 56 48 57 68²Stage I 1 (1.8%) 0 2 (3.5%) 2 (2.9%)²Stage II 2 (3.6%) 1 (2.1%) 6 (10.5%) 4 (5.9%)²Stage III 5 (8.9%) 3 (6.3%) 3 (5.3%) 4 (5.9%)²Stage IV 48 (85.7%) 44 (91.7%) 46 (80.7%) 58 (85.3%)²Time from²diagnosis to²randomization²(months)* 0.6620b²Number 57 49 60 69²Median 1.4 1.8 1.6 1.7²Mean (SD) 12.6 (30.9) 9.2 (16.9) 9.8 (19.8) 9.7 ²(17.5)²Min : Max 0 : 149 0 : 80 0 : 84 0 : 80²'comparing frequency distribution based on Fisher's exact test - 2-sided. ²busing ANOVA ²(type 3) with factors:BIOPOP, BIOPOP. Records with missing values for factors ²or ²response were excluded from statistical analyses.²Frequency distribution of covariates is compared between evaluable and non-²evaluable²populations²*If the day of initial date of diagnosis is missing, it is considered as the ²first day of the²month² Table 3: Summary of organs involved at baseline²Biomarkers non Biomarkers evaluable²evaluable population population p-²value²Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70) ²<DP=17>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²17²Biomarkers non Biomarkers evaluable²evaluable population population p-²value²Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70)²Number of²metastatic organs ²involved at²baseline (excluding²primary site) [n(%)] ²0.1711a²Number 57 49 60 70²0 0 0 1 (1.7%) 0² 1 16 (28.1%) 15 (30.6%) (25.0%) 12 (17.1%)²44²>1 41(71.9%) 34 (69.4%) (73.3%) 58 (82.9%)²Metastatic organs ²involved at²baseline (excluding²primary site) 59²[n(%)]* 57 (100%) 49 (100%) (98.3%) 70 (100%)²47²Liver 44 (77.2%) 42 (85.7%) (78.3%) 57 (81.4%)² Lung 27 (47.4%) 18 (36.7%) (41.7%) 28 (40.0%)² Lymph nodes 26 (45.6%) 25 (51.0%) (50.0%) 38 (54.3%)²14²Muscle / soft tissue 9(15.8%) 6(12.2%) (23.3%) 10(14.3%)²8²Peritoneum 8 (14.0%) 7 (14.3%) (13.3%) 16 (22.9%)²Pleura 5 (8.8%) 7 (14.3%) 2 (3.3%) 1 (1.4%)²Adrenal 2 (3.5%) 0 1 (1.7%) 1 (1.4%)²Bone 2 (3.5%) 1 (2.0%) 4 (6.7%) 4 (5.7%)²Kidneys 1 (1.8%) 0 0 0²Spleen 1 (1.8%) 1 (2.0%) 1 (1.7%) 1 (1.4%)²Bladder 0 0 1 (1.7%) 1 (1.4%)²<DP=18>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²18²Biomarkers non Biomarkers evaluable²evaluable population population p-²value²Aflibercept / ²Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=57) (N=49) (N=60) (N=70)²Metastatic organs ²involved at²baseline class ²(excluding primary²site) [n(%)] ²0.3536a²Number 57 49 60 70²No liver²metastasis, or²liver and other 51²metastases 49 (86.0%) 39 (79.6%) (85.0%) 63 ²(90.0%)²Liver metastasis 9²only 8 (14.0%) 10 (20.4%) (15.0%) 7 ²(10.0%) ²'comparing frequency distribution based on Fisher's exact test - 2-sided. ²Records with ²missing values for factors or response were excluded from statistical ²analyses.²Frequency distribution of covariates is compared between evaluable and non-²evaluable ² populations²*Percentages are not additive (sum greater than 100%)²Safety evaluation ² A. Extent of exposure ²Table 4 below shows that patients in the biomarkers evaluable population were ²exposed slightly longer to treatment than patients in the biomarkers non-²evaluable ²population (median number of cycles: 12 versus 9 or 10).²There was no difference in exposure between treatment arms in the biomarkers² evaluable population.²Table 4: Summary of overall study treatment exposure²Biomarkers non evaluable Biomarkers evaluable²population ²population²Aflibercept / ²Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=56) (N=49) (N=60) (N=70) ²<DP=19>²CA 02955302 2017-01-16²WO 2016 / 008975 ²PCT / EP2015 / 066262²19²Biomarkers non evaluable Biomarkers evaluable²population population² Aflibercept / Aflibercept / ²mFolfox6 mFolfox6 mFolfox6 mFolfox6²(N=56) (N=49) (N=60) (N=70)²Number of cycles ²received by patient²Sum 614 572 770.0 865.0²Mean (SD) 11.0 (7.0) 11.7 (9.5) ²12.8 (7.0) 12.4 (7.9)²Median 10.0 9.0 12.0 12.0²Min : Max 1 : 43 1 : 44 3 : 33 1 : ²42²Number of cycles ²received by patient²1 2 (3.6%) 3 (6.1%) 0 2 ²(2.9%)²2 2 (3.6%) 2 (4.1%) 0 2 ²(2.9%)²3 3 (5.4%) 1 (2.0%) 2 (3.3%) 2 ²(2.9%)²4 3 (5.4%) 4 (8.2%) 5 (8.3%) 8 ²(11.4%)² 1 (1.8%) 0 1 (1.7%) 2 (2.9%)²6 1 (1.8%) 6(12.2%) 0 4 ²(5.7%)²7 1 (1.8%) 5(10.2%) 1 (1.7%) 3 ²(4.3%)²8 6(10.7%) 1 (2.0%) 11(18.3%) 2 ²(2.9%)²9 6 (10.7%) 3 (6.1%) 0 ² 5 (7.1%)²5 (8.9%) 2 (4.1%) 4 (6.7%) 3 (4.3%)²11-15 16(28.6%) 11(22.4%) ²21(35.0%) 13(18.6%)²16-20 7 (12.5%) 4 (8.2%) 6 ²(10.0%) 14 (20.0%)²21-25 1 (1.8%) 3 (6.1%) 5 (8.3%) 6 ²(8.6%)²>25 2 (3.6%) 4 (8.2%) 4 (6.7%) 4 ²(5.7%)²Duration of²exposure (weeks)²Number 56 49 60 70²Mean (SD) 25.2 (16.0) 27.5 (22.4) 29.5 ²(16.4) 28.7 (18.7)²Median 24.1 23.1 27.3 25.4²Min : Max 2 : 95 2 : 106 6 : 77 2 : ²88 ²Duration of exposure = ((First date of last cycle + 14) - First date of first ²cycle) / 7 ²SD: standard deviation²B. Plasma profiling ²The plasma concentration of 27 cytokines, growth factors or soluble receptors ²was²5 determined by enzyme-linked immunosorbent assays (ELISA) using two ²Fluorokine ²MAP kits (the human angiogenesis panel A and the human high sensitivity ²cytokine panel;²<DP=20>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262² R&D Systems). Competition experiments were conducted to test interference of ²aflibercept with the detection of VEGF-A, VEGF-D and placental growth factor ²(PIGF). ²Angiopoietin-2 (ANGPT2), SDF1-a, HGF, VEGF-C, soluble VEGF receptor 3 (5FLT4, ²5VEGFR3) and sVEGFR2 were assessed by single ELISA (R&D Systems). Plasma²5 markers were analyzed at baseline, at 30 and 60 days after the first ²study treatment²infusion and 30 days after the last aflibercept infusion.²Statistical analysis ²Differences between patients with evaluable biomarkers and patients without ²evaluable ²biomarkers were assessed using a two-sided Fisher's exact test for categorical ²variables²10 and ANOVA for continuous variables. Biomarkers were analyzed as ²quantitative variables,²by coding the absence or presence of a somatic mutation as 0 or 1, and SNP ²genotypes ²as 0, 1 or 2 depending on the number of minor alleles present. The linear ²effects of ²baseline biomarkers on PFS were assessed using a Cox proportional hazard model ²with ²the following co-variates: Eastern Cooperative Oncology Group (ECOG) ²performance²15 status (0-1 versus 2), liver-only metastases (yes / no), and the number of ²distant metastasis²organs (1 versus >1), a treatment effect, a biomarker effect and a biomarker-²treatment ²interaction effect. The significance of the latter two effects was jointly ²tested by a two-²degrees-of-freedom Wald test. Extended statistical methods are described in ²supplementary methods.²20 Results ²Of the 236 patients in the ITT population of the AFFIRM trial 227 (96%) were ²evaluable for ²response. Of these, 130 (57%) provided at least one biological sample, 60 ²(46%) and 70 ²(54%) of which participated in the mFOLFOX6 and mFOLFOX6 plus aflibercept ²arms, ²respectively. There was no major difference at a false discovery rate (FDR)-²adjusted P-²value of 0.05 between patients who provided a biological sample and those who ²did not in²terms of patient biometrics, ethnicity, and disease characteristics at ²baseline, or at efficacy ²and safety endpoints (Table 1). Of those who provided at least one biological ²sample, 51 ²(39%) provided samples for each of the 3 biomarker types, with 88 (68%) and 97 ²(74%) ²patients providing samples for 2 or 1 of the biomarker types respectively. ²Each biomarker²type was analyzed separately, to avoid patient groups that were too small for ²sub-²analyses.²<DP=21>²CA 02955302 2017-01-16²WO 2016 / 008975 ²PCT / EP2015 / 066262²21²Profiling of plasma markers for efficacy²Plasma levels of 27 markers were measured at different time points (i.e., at ²baseline [87 ²patients]; 30 and 60 days after start of treatment [82 and 73 patients]; and ²30 days after² the last treatment [56 patients] as indicated on Table 5.²Table 5: Number of observations per time point - total, below limit of ²quantification ²(LOQ) and of detection (LOD)²EOT + 30²Baseline Day 30 Day 60 Days ²Total <LOQ <LOD Total <LOQ <LOD Total <LOQ <LOD Total <LOQ <LOD ²ANGPT1 87 1 1 80 1 0 73 0 0 56 0 0²ANGPT2 86 1 82 1 72 0 55 1²CSF2 84 28 17 80 38 16 72 32 17 54 25 16²CXCL12 86 0 0 82 2 2 72 0 0 55 0 0²FGF1 87 14 14 80 16 16 73 18 18 56 8 8²Endostatin 87 80 73 56²FGF2 87 19 11 80 20 15 73 19 11 56 10 5²FIGF 87 12 12 80 4 4 73 1 1 56 1 1²HGF 86 0 0 82 7 7 72 1 1 55 1 1²IFNG 84 6 4 80 8 3 72 6 2 54 11 8²11_10 84 0 0 80 1 1 72 2 2 54 0 0²1L12 84 3 3 80 1 1 72 3 3 54 8 7²11_113 84 1 1 80 0 0 72 1 1 54 4 4²1L2 84 3 2 80 3 1 72 1 1 54 8 6²1L4 84 2 2 80 1 1 72 2 2 54 7 7²1L5 84 2 1 80 3 0 72 1 0 54 1 1²1L6 84 0 0 80 2 1 72 0 0 54 1 1²1L8 84 0 0 80 1 1 72 0 0 54 0 0²PGF 87 8 8 80 6 6 73 2 2 56 1 1²TNF 84 2 1 80 2 1 72 1 0 54 2 1²PDGFA 87 80 73 56²VEGFA 84 6 1 80 4 1 72 2 0 54 4 0²PDGFB 87 80 73 56²VEGFC 86 1 1 82 0 0 72 0 0 55 1 0 ²sFLT4 86 0 0 82 7 7 72 1 1 55 1 1²THBS2 87 80 73 56²sKDR 86 0 0 82 7 7 72 1 1 55 1 1 ²<DP=22>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²22²All cytokines were measured in pg / ml, but some transformations were applied ²when ²necessary to obtain a symmetric distribution or smaller numerical values ²(ng / ml) for the ²association models, as depicted in table 6.²Table 6 - Selected transformations of original plasma cytokine levels (pg / ml)² ______________________________________________________________________ ²Cytokine Transformation²ANGPT1 log²ANGPT2 log²CSF2 square root²CXCL12 ng / ml²FGF1 cubic root²Endostatin log²FGF2 square root²FIGF cubic root²HGF Log²IFNG square root²ILI 0 cubic root²IL12 None²MB cubic root²IL2 square root²IL4 None²IL5 Log²IL6 Log²IL8 Log²PGF ng / ml²TNF square root²PDGFA log²VEGFA Log²PDGFB log²VEGFC Log²sFLT4 square root(ng / m1)²THBS2 log²sKDR square root(ng / m1)²We assessed the association of each plasma marker at baseline with PFS, while ²allowing ²for an interaction with treatment (Table 7). The lowest P-value was obtained ²for IL8 ²(P=0.0211; FDR=0.596 and P=0.0218 for interaction).²Table 7: Effect of baseline plasma biomarkers on PFS. The P-values associated ²to the²joint effect, the FDR-corrected joint effect, the plasma biomarker and the ²biomarker with ²treatment interaction effect are shown.²P-values²FDR-corrected Plasma level Treatment by²Protein Joint effect joint effect effect plasma level ²effect²1L8 0.0221 0.5962 0.6701 0.0218²THBS2 0.1408 0.6523 0.0545 0.2234²<DP=23>²CA 02955302 2017-01-16²WO 2016 / 008975 PCT / EP2015 / 066262²23²P-values²FDR-corrected Plasma level Treatment by²Protein Joint effect joint effect effect plasma level ²effect ²CXCL12 0.1610 0.6523 0.0573 0.1640²MO 0.1669 0.6523 0.0647 0.1311²Ang1 0.1768 0.6523 0.0724 0.0831²FIGF 0.1974 0.6523 0.6826 0.1704²FGF2 0.2063 0.6523 0.1722 0.0768²sVEGFR2 0.2171 0.6523 0.1616 0.0812²PDGFA 0.2474 0.6523 0.1155 0.1052²1L6 0.2689 0.6523 0.1061 0.1838²FGF1 0.3185 0.6523 0.4759 0.1536²VEGF-A 0.3202 0.6523 0.2549 0.7133²CSF2 0.3272 0.6523 0.4278 0.1679²1L12 0.3587 0.6523 0.3664 0.1808²IFNg 0.3855 0.6523 0.8662 0.2984²1L4 0.3866 0.6523 0.7326 0.4796²PIGF 0.5008 0.7955 0.3018 0.2595²PDGFB 0.5442 0.8163 0.4795 0.2963²IL1B 0.6025 0.8439 0.3183 0.3595²HGF 0.6286 0.8439 0.3369 0.5093²1L2 0.6564 0.8439 0.7696 0.4093²Endostatin 0.9021 0.9783 0.7429 0.9824²sFLT4 0.9063 0.9783 0.6849 0.8668²TNF 0.9461 0.9783 0.8736 0.7442²Ang2 0.9698 0.9783 0.9279 0.9747²VEGF-C 0.9772 0.9783 0.8375 0.8307²1L5 0.9783 0.9783 0.8813 0.8501 ²The Cox model assumes a linear relationship between the plasma marker and the ²log of the PFS hazard function, but since this hypothesis may be violated when ²analyzing²continuous markers that vary considerably, a threshold effect may be more ²relevant. We²explored this possibility by searching for the optimal cut-off level that ²maximizes the ²interaction with the treatment and the plasma marker. For IL8, the optimal cut-²off was at ²19 pg / ml (77th percentile). A model, in which IL8 was analyzed as a binary ²variable with ²this threshold, fitted better than a model with continuous IL8 levels (AIC of ²469.3 versus²477.6). Patients with low IL8 levels (19 pg / ml, 77% of patients) exhibited a ²longer PFS in²the aflibercept / mFLOFOX6 arm than in the mFOLFOX6 arm (Table 8).²<DP=24>²CA 02955302 2017-01-16²WO 2016 / 008975 ²PCT / EP2015 / 066262²24²Table 8: Kaplan-Meier estimates of effect of biomarkers on months of PFS.²mFOLFOX6 Aflibercept plus Hazard Ratio vs²mFOLFOX6 mFOLFOX6²Median (99% Cl)²Median (99% Cl) (99% Cl) ²Plasma protein markers ²All patients 8.8 (6.57-10.02) 8.5 (6.67-²10.05) 0.979 (0.505-1.897)²1L8 19pg / mL 8.8 (5.62-10.91) 9.3 (7.52-²11.10) 0.764 (0.363-1.607)²1L8> 19pg / mL 8.8 (5.09-15.64) 4.1 (2.33-8.54) 2.71 (0.735-²9.984) ²CI, confidence interval; mt, mutant; wt, wild-type.²We also analyzed whether treatment-related changes in plasma markers could ²predict²aflibercept treatment outcome. The Cox model included the effect of baseline ²plasma ²levels and the difference in expression measured at baseline and the last time ²point ²before discontinuation, disease progression or death (i.e., at 30 days or 60 ²days of ²treatment), while allowing for interaction with the treatment arm. IL8 was the ²only marker²with a significant effect of change from baseline on PFS (P=0.0018; ²FDR=0.0478; Table²9). This effect did not differ between treatment arms (P=0.2028). High ²baseline or post-²baseline increased 1L8 levels corresponded to a higher probability of disease ²progression ²at 12 months (Figures 1 and 2).²Table 9: Effect of plasma marker changes from baseline on PFS. P-values (and ²FDR)²of the joint effects of plasma marker and treatment by plasma marker ²interactions are²shown. P-values for the change from baseline and the interaction are also ²presented.²P-values²Joint Joint effect Change from Treatment ²by Change²Protein effect FDR-corrected baseline effect ²from baseline effect²1L8 0.0018 0.0478 0.0006 0.2028²11_10 0.0342 0.4525 0.5214 0.8204²VEGFA 0.0619 0.4525 0.0189 0.0704²CXCL12 0.0670 0.4525 0.1714 0.0318²CSF2 0.0855 0.4619 0.0266 0.1682²VEGFC 0.1127 0.5072 0.0742 0.9355²1L5 0.1886 0.7275 0.0684 0.1657²Endostatin 0.2418 0.7882 0.9988 0.2668²PDGFA 0.3092 0.7882 0.1508 0.9922²TNF 0.3209 0.7882 0.1770 0.1554²1L4 0.3211 0.7882 0.1938 0.7631²FGF2 0.3851 0.8191 0.6468 0.3957²sFLT4 0.3944 0.8191 0.2533 0.1729²THBS2 0.4677 0.8426 0.2387 0.6683²FGF1 0.4770 0.8426 0.4669 0.2242²<DP=25>²CA 02955302 2017-01-16²WO 2016 / 008975 ²PCT / EP2015 / 066262² P-values²Joint Joint effect Change from Treatment ²by Change²Protein effect FDR-corrected baseline effect ²from baseline effect²PGF 0.4993 0.8426 0.2501 0.2755²ANGPT2 0.5638 0.8924 0.3373 0.8394²11_1 B 0.6261 0.8924 0.3345 0.5414²PDGFB 0.6583 0.8924 0.3730 0.6009²1L12 0.6611 0.8924 0.7934 0.7744²1L2 0.7984 0.9482 0.9352 0.6027²sKDR 0.8375 0.9482 0.6009 0.5613²HGF 0.8383 0.9482 0.5804 0.5665²IFNG 0.8559 0.9482 0.6923 0.9101²ANGPT1 0.8814 0.9482 0.6269 0.8538²FIGF 0.9131 0.9482 0.6838 0.7528²1L6 0.9963 0.9963 0.9893 0.9594²When plasma biomarkers were measured at baseline only, IL8 had the most ²prominent ²effect on PFS, which was best described as a threshold effect with high ²circulating IL8 ²(IL8>19 pg / mL) associated with a shorter PFS in the aflibercept-treated ²patients.²5 When plasma biomarkers were measured at baseline and during treatment, ²high levels of²circulating IL8 at baseline together with increased levels of IL8 measured ²during treatment ²were significantly associated with reduced PFS (FDR=0.0478).²CONCLUSIONS ²10 We identified that high IL8 levels at baseline correlated with shorter ²survival times,²and patients with increasing levels of IL8 during treatment were more likely ²to progress. ²This suggests that patients with high IL8 levels, at baseline or during ²treatment, ²are at increased risk of disease progression during aflibercept therapy.²

Claims

<DP=1>²26²CLAIMS²1. Use of interleukin-8 (IL-8) as a biomarker for predicting the outcome of ²the ²treatment with aflibercept, or ziv-aflibercept of a patient suspected to ²suffer from a ²cancer.²2. A method of determining whether a patient suspected to suffer from cancer ²is a ²candidate for aflibercept, or ziv-aflibercept therapy for the said cancer ²comprising ²the step of subjecting a patient's biological sample to at least one assay to ²measure at baseline the IL-8 level, wherein when the biological sample IL-8 ²level ²is low relative to a reference level of expression of IL-8, the patient is ²identified as ²a candidate for therapy for cancer.²3. A method of determining whether a patient suspected to suffer from cancer ²is a ²candidate for aflibercept, or ziv-aflibercept therapy for the said cancer ²comprising ²the step of subjecting a patient's biological sample to at least one assay to ²measure at baseline the IL-8 level, wherein when the biological sample IL-8 ²level ²is high relative to a reference level of expression of IL-8, the patient is ²identified as ²not being a candidate for therapy for cancer.²4. A method for treating a patient with a cancer with aflibercept, or ziv-²aflibercept, ²comprising administering a therapeutically effective amount of aflibercept, or ²ziv-²aflibercept to the patient, wherein the IL-8 level in the patient's biological ²sample is ²lower than the reference level of expression of IL-8.²5. A method according to any of claims 2 to 4, wherein the relative to a ²reference ²level of expression of IL-8 is between around 10 and around 30 pg / ml.²6. A method according to any of claims 2 to 5, wherein the relative to a ²reference ²level of expression of IL-8 around 19 pg / ml.²7. A method of optimizing therapeutic efficacy for treatment of a cancer, ²comprising ²the steps of:².cndot. administering aflibercept, or ziv-aflibercept to a patient ²suspected to suffer from a ²cancer; and².cndot. determining the level of interleukin-8 (IL-8) in patient's ²biological sample,²<DP=2>²27²wherein an increase of the interleukin-8 (IL-8) level indicates the need to ²decrease the ²amount of aflibercept, or ziv-aflibercept in subsequent administrations.²8. A method of managing the risk to allow a safe use of aflibercept, or ziv-²aflibercept ²in the treatment of a patient suspected to suffer from a cancer, said method ²comprising the following steps:²(a) before beginning of the treatment with aflibercept, or ziv-aflibercept, ²determining the interleukin-8 (IL-8) level in a biological sample from the ²patient;²(b) along the treatment determining the interleukin-8 (IL-8) level in a ²biological ²sample from the patient²(c) comparing the interleukin-8 (IL-8) level determined in step (b) with the ²level ²determined in step (a),²whereby a higher interleukin-8 (IL-8) level in the sample of step (b) compared ²to ²that of level in the sample of step (a) indicates that the patient should be ²closely ²monitored.²9. A method according to any of claims 2 to 8, wherein the biological sample ²is ²chosen from the group consisting of blood, serum and plasma.²10. A method according to any of claims 2 to 8, wherein the cancer is colon ²cancer, a ²colorectal cancer or a rectal cancer.²11.A method according to claim 10 wherein the colorectal cancer is a ²metastatic ²colorectal cancer.²12.A method according to any of claims 2 to 8, wherein the interleukin-8 (IL-²8) level ²which is determined is the circulating level.²13.A method according to any of claims 4 to 6, said method comprising ²administering ²to said patient therapeutically effective amounts of aflibercept, or ziv-²aflibercept, ²oxaliplatin, 5-fluorouracil (5-FU) and folinic acid.²14.A method according to any of claims 4 to 6, said method comprising ²administering ²to said patient therapeutically effective amounts of aflibercept, or ziv-²aflibercept, ²folinic acid, 5- fluorouracil (5-FU) and irinocetan.²15.A method according to any one of claims 4 to 6, wherein said patient has ²previously been treated with therapy based on oxaliplatin or on bevacizumab.²<DP=3>²28²16.A method according to any one of claims 4 to 6, wherein said patient has ²failed ²with chemotherapy, radiotherapy or surgery.²17.A method according to any one of claims 4 to 6, wherein folinic acid at a ²dosage ²comprised between about 200 mg / m2 and about 600 mg / m2, 5-fluorouracil (5-FU) ²at a dosage comprised between about 2000 mg / m2 and about 4000 mg / m2, ²irinocetan at a dosage comprised between about 100 mg / m2 and about 300 mg / m2 ²and aflibercept at a dosage comprised between about 1 mg / kg and about 10 ²mg / kg are administered to patient.²18.A method according to any one of claims 4 to 6 and 17, wherein folinic acid ²at a ²dosage of about 400 mg / m2, 5-fluorouracil (5-FU) at a dosage of about 2800 ²mg / m2, irinocetan at a dosage of about 180 mg / m2 and aflibercept at a dosage ²of ²about 4 mg / kg are administered to patient.²19.A method according to any one of claims 4 to 6, 17 and 18 wherein folinic ²acid is ²administered intravenously at a dosage of about 400 mg / m2, 5-fluorouracil (5-²FU) ²is administered intravenously at a dosage of about 2800 mg / m2, irinocetan is ²administered intravenously at a dosage of about 180 mg / m2 and aflibercept is ²administered intravenously at a dosage of about 4 mg / kg and wherein the ²combination is administered every two weeks.²20.A method according to any one of claims 4 to 6 and 17 to 19, wherein the ²folinic ²acid, 5- fluorouracil (5-FU), irinocetan and aflibercept are administered ²intravenously every two weeks for a period comprised between 9 and 18 weeks.²21.A method according to any one of claims 4 to 6 and 17 to 20, wherein the ²folinic ²acid is administered intravenously immediately after aflibercept ²administration.²22. A method according to any one of claims claims 4 to 6 and 17 to 21, ²wherein the ²folinic acid is administered intravenously immediately after aflibercept ²administration over a period of about 2 hours.²23. Aflibercept, or ziv-aflibercept for treating a patient suspected to suffer ²from cancer, ²wherein the IL-8 level in the patient's biological sample is lower than the ²reference ²level of expression of IL-8.²24.A kit for predicting whether a patient suspected to suffer from cancer is a ²candidate ²for aflibercept, or ziv-aflibercept therapy, which kit comprises:²<DP=4>²29²a) means for measuring the interleukin-8 (IL-8) level; and²b) optionally, a label giving instructions for the use of said kit in ²predicting ²whether a a patient suspected to suffer from cancer is a candidate for ²aflibercept, or ziv-aflibercept therapy.²25. An article of manufacture comprising:²a) a packaging material;²b) means for measuring the interleukin-8 (IL-8) level; and²c) a label giving instructions for the use of said kit in predicting whether a ²a ²patient suspected to suffer from cancer is a candidate for aflibercept, or ²ziv-aflibercept therapy.²