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:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of International Application No. PCT/EP2015/066262, filed Jul. 16, 2015, which claims priority to EP Application No. 14306172.9, filed Jul. 18, 2014. 
     
    
     BACKGROUND OF THE INVENTION 
     Field of the Invention 
       [0002]    The present invention concerns the use of interleukin 8 (IL8) as a biomarker for predicting the outcome of the treatment with aflibercept, or ziv-aflibercept, of a patient suspected to suffer from cancer. 
         [0003]    Aflibercept, or ziv-aflibercept, also referred to as VEGFR1 R2-Fc.DELTA.C1 FIt1D2.FIk1D3.Fc.DELTA.C1 or AVE0005, is a homo dimer protein, with each dimer comprising two identical monomers, each of which is a fusion protein comprising the signal sequence of VEGFR1 fused to the D2 Ig domain of the VEGFR1 receptor, itself fused to the D3 Ig domain of the VEGFR2 receptor, in turn fused to the Fc domain of IgG1. 
         [0004]    The protein chain is glycosylated, with N-acetyl-glucosamine, fucose, galactose, mannose and sialic acids contributing to the carbohydrate structures. The N-linked oligosaccharides consist of mainly bi-antennary structures with zero, one or two terminal sialic acids. The monomer has the amino acid sequence SEQ ID No 1. 
         [0005]    The U.S. Food and Drug Administration (FDA) already approved aflibercept under the trade name EYLEA® for the treatment of patients with neovascular (wet) age-related macular degeneration (AMD). In particular, EYLEA® is the trade name for aflibercept as generated, processed and formulated for intravitreal injection. 
         [0006]    At the time of registration of aflibercept (zaltraP) for cancer indication, and In light of aflibercept&#39;s approved use in treating AMD, the FDA requested that a different name (ziv-aflibercept) be given for the compound&#39;s use in the treatment of cancer. Thus, ziv-aflibercept is the United States Adopted Name (USAN) accepted by FDA to designate a pharmaceutical composition comprising aflibercept as generated, processed and formulated for injection via intravenous infusion. Ziv-aflibercept has been approved by the FDA for sale under the tradename ZALTRAP® for the treatment of metastatic colorectal cancer (mCRC). 
         [0007]    The European Medicines Agency (EMA) approved zaltrap as well however did not request separate names for the compound. Thus, in the European Union the name “aflibercept” is used regardless of the indication. 
         [0008]    ZALTRAP® and EYLEA® are obtained by slightly different processes. They both contain aflibercept or ziv-aflibercept, but the ratio of aggregates of aflibercept or ziv-aflibercept is slightly different in ZALTRAP® and EYLEA®. 
         [0009]    ZALTRAP® approval was based on data obtained from the VELOUR trial—a multicenter, randomized, placebo-controlled phase III trial, which compared the efficacy of aflibercept versus placebo in combination with the FOLFIRI regimen for patients with mCRC previously treated with an oxaliplatin containing regimen. 
         [0010]    AFFIRM, an open-label, non-comparative, phase II study, was conducted to assess the combination of aflibercept and modified FOLFOX6 (mFOLFOX6) given as first-line therapy in patients with mCRC. The primary endpoint was 12-month progression-free survival (PFS) whereas exploration of biomarkers was among the secondary objectives. 
         [0011]    In an attempt to understand the key factors associated with aflibercept efficacy and safety, an investigation assessing biomarkers for aflibercept treatment in prospectively collected, tumor tissues and serially sampled plasma from patients participating in the AFFIRM study was conducted. 
         [0012]    Plasma proteins and genetic variants, representing either single nucleotide polymorphisms (SNPs) in angiogenicpathway genes or somatic mutations in key oncogenic drivers of mCRC, were analyzed to assess if they could predict response to aflibercept with respect to PFS. Subsequently, one also assessed whether any of these markers correlated with anti-angiogenic drug-induced AEs, such as gastrointestinal perforation, thrombosis, hypertension and proteinuria. 
         [0013]    Despite the efficacy and the safety of the treatment of cancer by aflibercept it remains a goal to better identify patients who should benefit more from the treatment. 
         [0014]    It has now been discovered that high IL8 levels at baseline correlated with shorter survival times, and patients with increasing levels of IL8 during treatment were more likely to progress. This suggests that patients with high IL8 levels, at baseline or during treatment, are at increased risk of disease progression during aflibercept therapy. 
         [0015]    The role of IL8-in tumor development and progression is suggested in the prior art 
         [0016]    In a phase II trial hepatocellular carcinoma patients received bevacizumab (Boige V, Malka D, Bourredjem A et al. Efficacy, safety, and biomarkers of single-agent bevacizumab therapy in patients with advanced hepatocellular carcinoma. Oncologist 2012; 17: 1063-1072). Circulating endothelial cells (CECs) and plasma cytokines and angiogenic factors (CAFs) were measured at baseline and throughout treatment. 
         [0017]    This study showed that elevated baseline IL-8 (above 80 pg/ml) and IL-6 levels were correlated with both a shorter progression-free survival (PFS) interval and a shorter overall survival (OS) time. 
         [0018]    However the authors cite another study with sorafenib wherein conversely, IL-8 was the only serum cytokine not correlated with the PFS outcome. 
         [0019]    In another phase II trial metastatic colorectal cancer (mCRC) patients received bevacizumab (Kopetz S, Hoff P M, Morris J S et al. Phase II trial of infusional fluorouracil, irinotecan, and bevacizumab for metastatic colorectal cancer: efficacy and circulating angiogenic biomarkers associated with therapeutic resistance. J Clin Oncol 2010; 28: 453-459). 
         [0020]    Levels of 37 CAFs were assessed at baseline, during treatment, and at the time of progressive disease (PD). 
         [0021]    The authors conclude that elevated baseline IL8 above the median value of 3.7 pg/mL was associated with shorter PFS times. 
         [0022]    These results were obtained with bevacizumab which is an antibody. Aflibercept is not an antibody but a fusion protein and thus has a different mode of action. 
         [0023]    It is not possible to predict the effect of a biomarker on the efficiency of cancer treatment by a given drug from results obtained with another drug. 
         [0024]    A fortiori the threshold above or under which a patient is considered as a candidate for treatment cannot be extrapolated between two different drugs. 
       BRIEF SUMMARY OF THE INVENTION 
       [0025]    The invention relates to 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. 
         [0026]    In one aspect, the present invention provides a method of determining whether a patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-aflibercept therapy for the said cancer comprising the step of subjecting a patient&#39;s biological sample to at least one assay to measure at baseline the IL-8 level, wherein when the biological sample IL-8 level is low relative to a reference level of expression of IL-8, the patient is identified as a candidate for therapy for cancer. 
         [0027]    In another aspect, the present invention provides a method of determining whether a patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-aflibercept therapy for the said cancer comprising the step of subjecting a patient&#39;s biological sample to at least one assay to measure at baseline the IL-8 level, wherein when the biological sample IL-8 level is high relative to a reference level of expression of IL-8, the patient is identified as not being a candidate for therapy for cancer. 
         [0028]    In an embodiment the reference level of expression of IL-8 is comprised between around 10 and around 30 pg/ml. 
         [0029]    In a further embodiment the reference level of expression of IL-8 is comprised between around 15 and around 25 pg/ml or around 17 and around 21 pg/ml. 
         [0030]    In a further embodiment the reference level of expression of IL-8 is around 18 pg/ml, around 19 pg/ml or around 20 pg/ml. 
         [0031]    The invention relates also to a method for treating a patient with a cancer with aflibercept, or ziv-aflibercept, comprising administering a therapeutically effective amount of aflibercept, or ziv-aflibercept to the patient, wherein the IL-8 level in the patient&#39;s biological sample is low relative to a reference level of expression of IL-8. 
         [0032]    The invention further relates to a method of optimizing therapeutic efficacy for treatment of a cancer, comprising the steps of:
       a) administering aflibercept, or ziv-aflibercept to a patient suspected to suffer from a cancer; and   b) determining the level of interleukin-8 (IL-8) in patient&#39;s biological sample,
 
wherein an increase of the interleukin-8 (IL-8) level indicates the need to decrease the amount of aflibercept, or ziv-aflibercept in subsequent administrations.
       
 
         [0035]    A further object of the invention is a method of managing the risk to allow a safe use of aflibercept, or ziv-aflibercept in the treatment of a patient suspected to suffer from a cancer, said method comprising the following steps:
       a) before beginning of the treatment with aflibercept, or ziv-aflibercept, determining the interleukin-8 (IL-8) level in a biological sample from the patient;   b) along the treatment determining the interleukin-8 (IL-8) level in a biological sample from the patient   c) comparing the interleukin-8 (IL-8) level determined in step (b) with the level determined in step (a),
 
whereby a higher interleukin-8 (IL-8) level in the sample of step (b) compared to that of level in the sample of step (a) indicates that the patient should be closely monitored.
       
 
         [0039]    In an embodiment of one of the methods described above the biological sample is chosen from the group consisting of blood, serum and plasma. 
         [0040]    In an embodiment of one of the methods described above the cancer is a colon cancer, a colorectal cancer or a rectal cancer. 
         [0041]    In a further embodiment of one of the the colorectal cancer is a metastatic colorectal cancer. 
         [0042]    In another embodiment of the invention, the subject is treated with aflibercept and further undergoes a chemotherapeutic treatment with oxaliplatin, 5-fluorouracil (5-FU) and folinic acid (i.e. the FOLFOX treatment), folinic acid, 5-fluorouracil and irinotecan (i.e. the FOLFIRI treatment), or 5-fluorouracil and folinic acid (i.e. the FUFOL or LV5FU2 treatment). 
         [0043]    The chemotherapeutic treatment may combine at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 agents, such as e.g. a combination of oxaliplatin, 5-fluorouracil (5-FU) and folinic acid (i.e. the FOLFOX treatment or the modified FOLFOX6 treatment as described in the example below), a combination of folinic acid, 5-fluorouracil and irinotecan (i.e. the FOLFIRI treatment), or a combination of 5-fluorouracil and folinic acid (i.e. the FUFOL or LV5FU2 treatment). 
         [0044]    In this regard the application WO2012146610 relates to a method of treatment of the mCRC by aflibercept, or ziv-aflibercept in combination with FOLFIRI. The content of this application is incorporated by reference. 
         [0045]    In an embodiment of one of the methods described above therapeutically effective amounts of aflibercept, or ziv-aflibercept, oxaliplatin, 5-fluorouracil (5-FU) and folinic acid are administered to said patient. 
         [0046]    In an embodiment of one of the methods described above therapeutically effective amounts of aflibercept, or ziv-aflibercept, folinic acid, 5-fluorouracil (5-FU) and irinotecan are administered to said patient. 
         [0047]    In a further embodiment of one of the methods described above folinic acid at a dosage comprised between about 200 mg/m 2  and about 600 mg/m 2 , 5-fluorouracil (5-FU) at a dosage comprised between about 2000 mg/m 2  and about 4000 mg/m 2 , irinotecan at a dosage comprised between about 100 mg/m 2  and about 300 mg/m 2  and aflibercept at a dosage comprised between about 1 mg/kg and about 10 mg/kg are administered to patient. 
         [0048]    In a further embodiment of one of the methods described above folinic acid at a dosage of about 400 mg/m 2 , 5-fluorouracil (5-FU) at a dosage of about 2800 mg/m 2 , irinotecan at a dosage of about 180 mg/m 2  and aflibercept at a dosage of about 4 mg/kg are administered to patient. 
         [0049]    In a further embodiment of one of the methods described above folinic acid is administered intravenously at a dosage of about 400 mg/m 2 , 5-fluorouracil (5-FU) is administered intravenously at a dosage of about 2800 mg/m 2 , irinotecan is administered intravenously at a dosage of about 180 mg/m 2  and aflibercept is administered intravenously at a dosage of about 4 mg/kg and wherein the combination is administered every two weeks. 
         [0050]    In a further embodiment of one of the methods described above folinic acid, 5-fluorouracil (5-FU), irinotecan and aflibercept are administered intravenously every two weeks for a period comprised between 9 and 18 weeks. 
         [0051]    In a further embodiment of one of the methods described above folinic acid is administered intravenously immediately after aflibercept administration. It can be also administered intravenously immediately after aflibercept administration over a period of about 2 hours. 
         [0052]    In a further embodiment of one of the methods described above irinotecan is administered intravenously immediately after aflibercept administration. It can be also administered intravenously immediately after aflibercept administration over a period of about 90 minutes. 
         [0053]    In a further embodiment of one of the methods described above 5-fluorouracil (5-FU) is administered immediately after aflibercept administration. 
         [0054]    In a further embodiment of one of the methods described above a first quantity of 5-fluorouracil (5-FU) is administered intravenously immediately after aflibercept administration and a second quantity of 5-FU is administered intravenously after the first quantity in continous infusion. 
         [0055]    In a further embodiment of one of the methods described above about 400 mg/m 2  of 5-fluorouracil (5-FU) is administered intravenously over a period of 2 to 4 minutes after aflibercept administration and wherein 2400 mg/m 2  of 5-FU is administered intravenously over around 46 hours after the administration of the 400 mg/m 2  in continous infusion. 
         [0056]    In an embodiment said patient has previously been treated with therapy based on oxaliplatin or on bevacizumab. 
         [0057]    In another embodiment said patient has failed with chemotherapy, radiotherapy or surgery. 
         [0058]    The invention relates also to aflibercept, or ziv-aflibercept for treating a patient suspected to suffer from cancer, wherein the IL-8 level in the patient&#39;s biological sample is lower than between around 15 and around 50 pg/ml. 
         [0059]    The invention further relates to a kit for predicting whether a patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-aflibercept therapy, which kit comprises:
       a) means for measuring the interleukin-8 (IL-8) level; and   b) Optionally, a label giving instructions for the use of said kit in predicting whether a patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-aflibercept therapy.       
 
         [0062]    Another aspect of the invention further relates to an article of manufacture comprising:
       a) a packaging material;   b) means for measuring the interleukin-8 (IL-8) level; and   c) a label giving instructions for the use of said kit in predicting whether a patient suspected to suffer from cancer is a candidate for aflibercept, or ziv-aflibercept therapy.       
 
         [0066]    The above methods and use of the invention may be, for instance, in vitro or ex vivo methods and use. 
         [0067]    Means for measuring the expression level of IL8 protein are well-known in the art and include immunoassay such as ELISA assay. The means for measuring IL8 protein include antibodies specifically binding to IL8. Such means can be labeled with detectable compound such as fluorophores or radioactive compounds. For example, the probe or the antibody specifically binding to IL8 may be labeled with a detectable compound. Alternatively, when the kit comprises an antibody, the kit may further comprise a secondary antibody, labeled with a detectable compound, which binds to an unlabeled antibody specifically binding to IL8. 
         [0068]    The means for measuring the expression level of IL8 may also include reagents such as e.g. reaction and/or washing buffers. The means may be present, e.g., in vials or microtiter plates, or be attached to a solid support such as a microarray as can be the case for primers and probes. 
         [0069]    Aflibercept, or ziv-aflibercept is provided in a formulation which is not prejudicial to the patient to be treated. 
         [0070]    In an embodiment aflibercept, or ziv-aflibercept is provided in a formulation with sucrose and polysorbate 20 (stabilisers), sodium chloride, citrate buffer, and sodium phosphate buffer, adjusted to final pH. 
         [0071]    In another embodiment aflibercept, or ziv-aflibercept, is supplied in two drug product presentations:
   a presentation at 100 mg aflibercept, or ziv-aflibercept/4.0 mL (nominal concentration).   a second presentation at 200 mg aflibercept, or ziv-aflibercept/8.0 mL (nominal concentration).   
 
         [0074]    Both presentations are manufactured from the same bulk sterile solution at 25 mg/mL of aflibercept, or ziv-aflibercept. 
         [0075]    Prior to infusion to the patient, the concentrate solution is diluted with 0.9% sodium chloride solution or 5% dextrose. 
         [0076]    The anti-cancer agents used in the above recited method or use are provided in a pharmaceutically acceptable carrier, excipient or diluent which is not prejudicial to the patient to be treated. 
         [0077]    Pharmaceutically acceptable carriers and excipient that may be used in the compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminium stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-a-tocopherol polyethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. 
         [0078]    As appreciated by skilled artisans, compositions are suitably formulated to be compatible with the intended route of administration. Examples of suitable routes of administration include parenteral route, including for instance intramuscular, subcutaneous, intravenous, intraperitoneal or local intratumoral injections. The oral route can also be used, provided that the composition is in a form suitable for oral administration, able to protect the active principle from the gastric and intestinal enzymes. 
         [0079]    The terms “Therapy”, “therapeutic”, “treatment” and “treating” are used herein to characterize a therapeutic method or process that is aimed at (1) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease state or condition to which such term applies; (2) alleviating or bringing about ameliorations of the symptoms of the disease state or condition to which such term applies; and/or (3) reversing or curing the disease state or condition to which such term applies. 
         [0080]    As used herein, the terms “interleukin-8” and “IL-8” are used interchangeably and refer to all of the naturally-occurring isoforms, including alternative splice variants, allelic variants and include naturally occurring variants, SNPs (single nucleotide polymorphisms), and truncated or secreted forms of IL-8 protein. 
         [0081]    In particular, the terms “interleukin 8” refers to the polypeptide comprising or consisting of the amino acid sequence corresponding to the UniProtKB/Swiss-Prot accession number P10145 (SEQ ID NO: 2) and/or
       a) a polypeptide corresponding to the mature isoform of the polypeptide of (a) (i.e. obtained after cleavage of the signal peptide); and/or   b) an allelic variant of the polypeptide of (a) or (b); and/or   c) a splice variant of a polypeptide of (a), (b) or (c); and/or   d) a constitutively active mutant of a polypeptide of (a), (b), (c) or (d).   e) an isoform obtained by proteolytic processing of a polypeptide of (a), (b), (c), (d) or (e).       
 
         [0087]    By “isoform of a polypeptide” is meant a polypeptide that has at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity to a full-length polypeptide reference sequence and has the same biological activity. In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. The &lt;&lt;needle&gt;&gt; program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may for example be used. The needle program is for example available on the ebi.ac.uk World Wide Web site. The percentage of identity in accordance with the invention can be calculated using the EMBOSS::needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix. 
         [0088]    “Isoform” also refers to all post-translationally modified forms of IL8 protein. Post-translationally modified isoforms may include acetylated, formylated, lipoylated, myristoylated, palm itoylated, alkylated, methylated, am idated, glycosylated, hyrdroxylated, nitrosylated, phosphorylated, sulphated, polysialylated and sialylated forms. 
         [0089]    The “reference level of expression of IL-8” may be determined as a single value or a range of values which is determined based on the expression level of IL-8 measured, for instance, in a population of healthy subjects or in a population of subjects in need of an aflibercept therapy. 
         [0090]    In an embodiment the reference level of expression of IL-8 is determined based on the expression level of IL-8 measured in a population of subjects in need of a aflibercept therapy. 
         [0091]    Typically, the analysed population could be divided into percentiles based on the measured level of expression of IL-8. The reference level could be defined as the percentile that provides the best separation between patients suffering from a cancer on which the treatment with aflibercept is efficient and patients suffering from a cancer on which the treatment with aflibercept is not efficient enough to cure it. 
         [0092]    In the study reported in Example 1 below, the reference level of expression of IL-8 was 19 pg/ml (77th percentile). 
         [0093]    However, the reference level of expression of IL-8 may vary i) according to the size of the studied population, and ii) depending on the method used for measuring the IL-8 expression. 
         [0094]    The level of interleukin 8 protein may be, for instance, determined using immunological detection methods such as an ELISA assay. The methods involve an antibody which binds to interleukin 8 protein, for example a monoclonal or polyclonal antibody, an antibody variant or fragments such as a single chain antibody, a diabody, a minibody, a single chain Fv fragment (sc(Fv)), a Sc(Fv)2 antibody, a Fab fragment or a F(ab′)2 fragment, or a single domain antibody. Such antibodies are well known in the art and are commercially available. They may also notably be obtained by immunization of animals (for example rabbits, rats or mice) with interleukin 8 protein. Antibodies may be used to determine protein expression in a range of immunological assays including competitive and non-competitive assay systems using techniques such as western blotting, immunohistochemistry/immunofluorescence (i.e protein detection on fixed cells or tissues), radioimmunoassay such as RIA (radio-linked immunoassay), ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays, ECLIA (electrochemiluminescence immunoassay) and protein A immunoassays. Such assays are routine and well known to the person skilled in the art (Ausubel et al (1994)  Current Protocols in Molecular Biology , Vol. 1, John Wiley &amp; Sons, Inc., New York). 
         [0095]    Protein expression of interleukin 8 may also be determined by proteomic method such as mass spectrometry assays (LC-MS or LC-MS/MS). Qualitative and quantitative mass spectrometric techniques are known and used in the art. To this aim, target peptides specific for marker proteins are selected and quantified based on calibration curves established with synthetic peptides labeled with stable isotopes. Enzymatic digests, spiked with a defined amount of isotope labeled target peptides, are analyzed by liquid chromatography coupled with mass spectrometry. The ratio between labeled and non-labeled target peptides is measured to assess target peptide concentrations and therefore protein marker concentration. 
         [0096]    The expression “circulating IL8” is intended to mean the IL8 proteins present in blood, serum and plasma. 
         [0097]    A “subject” or a “patient” may be a human or a non-human mammal, such as monkeys, dogs, cats, guinea pigs, hamsters, rabbits, cows, horses, goats and sheep. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS 
         [0098]      FIGS. 1 and 2  illustrate the relation between IL8 levels and the probability of disease progression. Depicted is the probability of disease progression after 12 months in relation to IL-8 plasma levels at baseline ( FIG. 1 ) and the difference between IL8 plasma levels at baseline and the last measurement point before disease progression ( FIG. 2 ). Briefly,  FIG. 1  shows that high IL8 levels correlate with increased probability for disease progression and that this effect is slightly more pronounced in aflibercept versus FOLFOX treated patients. On the other hand,  FIG. 2  shows that the increase in IL8 relative to baseline also corresponds to disease progression. Since increases in IL8 are plotted on a logarithmic scale, values between -3 and 0 correspond to an increase in IL8&lt;1 pg/mL, whereas values between 0 and 3 represent increases &gt;1 pg/mL. Data thus show that even small increases in IL8 relative to baseline already correspond to an increased probability of disease progression in the aflibercept arm. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLE: Effect of Interleukin 8 on PFS in the AFFIRM Study 
     Study ECF10668 (AFFIRM) 
       [0099]    EFC10668 was designed as a randomized, multinational, study comparing the adverse effects occurrence in patient with metastatic colorectal cancer (MCRC) treated with: 
         [0100]    i) a modified FOLFOX6 (a combination of oxaliplatin, 5-fluorouracil (5-FU) and folinic acid) given intravenously every 2 weeks as first-line treatment (arm A); or 
         [0101]    ii) aflibercept at 4 mg/kg combined with a modified FOLFOX6 given intravenously every 2 weeks as first-line treatment; or 
         [0102]    Schedule of Administration 
         [0103]    Patients were administered intravenously either with aflibercept immediately followed by oxaliplatin, 5-fluorouracil (5-FU) and folinic acid (modified FOLFOX6 regimen) or modified FOLFOX6 alone , depending on arm to which they were assigned, 
         [0104]    This treatment was repeated every 2 weeks until progression (or unacceptable toxicity, or consent withdrawal). 
         [0105]    Dosage 
         [0106]    The patients randomized in the aflibercept arm received 4 mg/kg IV every 2 weeks. 
         [0107]    The following were administered to patients in both treatment groups:
       Oxaliplatin (Eloxatin®)   olinic acid (also known as leucovorin)   5-fluorouracil       
 
         [0111]    Formulations of oxaliplatin, 5-fluorouracil, and folinic acid:
       Products used were those available in the hospital/clinic pharmacy   Route of administration: IV       
 
         [0114]    Dose: Oxaliplatin, folinic acid, and 5-fluorouracil were administered according to an mFOLFOX6 regimen, as follows:
       Oxaliplatin 85 mg/m 2  as a 2-hour IV infusion on day 1   Folinic acid 350 mg/m 2  as a 2-hour IV infusion on day 1   5-fluorouracil 400 mg/m 2  as an IV bolus on day 1, and then 2400 mg/m 2  as a 46-hour continuous IV infusion starting on day 1       
 
         [0118]    In case of body surface area &gt;2.0 m 2 , the actual doses of oxaliplatin and of 5-FU were to be adjusted to a maximum BSA of 2.0 m 2  for safety reasons. Dose reduction and/or treatment delay and/or treatment discontinuation were planned in case of severe toxicity. The modified FOLFOX6 regimen was administered after administration of aflibercept. 
         [0119]    Duration of Treatment: 
         [0120]    Treatment for an individual patient was administered up until progression or until unacceptable toxicity occurred or the patient withdrew consent. 
         [0121]    Treatment duration was estimated to be approximately 12 months. 
         [0122]    Demographics and Baseline Characteristics 
         [0123]    Table 1 below compares demographics and patient characteristics at baseline between biomarkers evaluable and non-evaluable populations. 
         [0124]    The “biomarkers evaluable population” is defined as the population of patients who provided a blood/tumor sample for biomarker assessment; while the “biomarkers non evaluable population” corresponds to patients who did not provide blood/tumor sample for biomarker assessment (e.g. patients who did not consent to biomarker study). 
         [0125]    All characteristics are similar between populations, except for the region of origin of the patients: Eastern Europe tends to be over-represented and other countries tend to be under-represented in the biomarkers evaluable population compared to the biomarker non evaluable population. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Summary of patient demographics and patient characteristics 
               
               
                 at baseline - Evaluable population 
               
             
          
           
               
                   
                 Biomarkers non 
                 Biomarkers 
                   
               
               
                   
                 evaluable population 
                 evaluable population 
               
             
          
           
               
                   
                   
                 Aflibercept/ 
                   
                 Aflibercept/ 
                   
               
               
                   
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
               
               
                   
                 (N = 57) 
                 (N = 49) 
                 (N = 60) 
                 (N = 70) 
                 p-value 
               
               
                   
                   
               
             
          
           
               
                 Gender [n(%)] 
                   
                   
                   
                   
                 1.0000 a   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
             
          
           
               
                 Male 
                 32 
                 (56.1%) 
                 33 
                 (67.3%) 
                 36 
                 (60.0%) 
                 43 
                 (61.4%) 
                   
               
               
                 Female 
                 25 
                 (43.9%) 
                 16 
                 (32.7%) 
                 24 
                 (40.0%) 
                 27 
                 (38.6%) 
               
             
          
           
               
                 Age (Years) 
                   
                   
                   
                   
                 0.2811 b   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
               
                 Median 
                     66.0 
                     62.0 
                     62.0 
                     62.5 
               
             
          
           
               
                 Mean (SD) 
                 63.7 
                 (10.0) 
                 61.8 
                 (9.5) 
                 61.3 
                 (9.4) 
                 61.7 
                 (8.7) 
                   
               
             
          
           
               
                 Min:Max 
                 44:87 
                 29:75 
                 37:81 
                 41:79 
                   
               
               
                 Age class [n(%)] 
                   
                   
                   
                   
                 0.2421 a   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
             
          
           
               
                  &lt;65 
                 27 
                 (47.4%) 
                 28 
                 (57.1%) 
                 38 
                 (63.3%) 
                 42 
                 (60.0%) 
                   
               
               
                 ≧65 but &lt;75 
                 23 
                 (40.4%) 
                 20 
                 (40.8%) 
                 20 
                 (33.3%) 
                 25 
                 (35.7%) 
               
               
                 ≧75 
                 7 
                 (12.3%) 
                 1 
                 (2.0%) 
                 2 
                 (3.3%) 
                 3 
                 (4.3%) 
               
             
          
           
               
                 Race [n(%)] 
                   
                   
                   
                   
                 0.0640 a   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
             
          
           
               
                 Caucasian/White 
                 41 
                 (71.9%) 
                 37 
                 (75.5%) 
                 49 
                 (81.7%) 
                 60 
                 (85.7%) 
                   
               
             
          
           
               
                 Black 
                  0 
                  0 
                  0 
                 1 
                 (1.4%) 
                   
               
             
          
           
               
                 Asian/Oriental 
                 16 
                 (28.1%) 
                 11 
                 (22.4%) 
                 11 
                 (18.3%) 
                 9 
                 (12.9%) 
                   
               
             
          
           
               
                 Other 
                  0 
                 1 
                 (2.0%) 
                  0 
                  0 
                   
               
             
          
           
               
                 Region 
                   
                   
                   
                   
                 0.0029 a   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
             
          
           
               
                 Western Europe 
                 37 
                 (64.9%) 
                 30 
                 (61.2%) 
                 37 
                 (61.7%) 
                 45 
                 (64.3%) 
                   
               
               
                 Eastern Europe 
                 3 
                 (5.3%) 
                 3 
                 (6.1%) 
                 12 
                 (20.0%) 
                 12 
                 (17.1%) 
               
               
                 Other countries 
                 17 
                 (29.8%) 
                 16 
                 (32.7%) 
                 11 
                 (18.3%) 
                 13 
                 (18.6%) 
               
             
          
           
               
                 BSA (m2) 
                   
                   
                   
                   
                 0.1091 b   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
               
                 Median 
                   1.8 
                   1.8 
                   1.8 
                   1.8 
               
             
          
           
               
                 Mean (SD) 
                 1.7 
                 (0.2) 
                 1.8 
                 (0.2) 
                 1.8 
                 (0.2) 
                 1.8 
                 (0.2) 
                   
               
             
          
           
               
                 Min:Max 
                 1:2 
                 1:2 
                 1:2 
                 1:2 
                   
               
               
                 Weight (kg) 
                   
                   
                   
                   
                 0.0838 b   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
               
                 Median 
                     67.6 
                     70.0 
                     73.2 
                     71.4 
               
             
          
           
               
                 Mean (SD) 
                 67.3 
                 (14.1) 
                 71.1 
                 (16.6) 
                 74.0 
                 (17.0) 
                 71.5 
                 (15.6) 
                   
               
             
          
           
               
                 Min:Max 
                  40:107 
                  40:115 
                  48:134 
                  40:117 
               
               
                   
               
               
                   a comparing frequency distribution based on Fisher&#39;s exact test - 2-sided. 
               
               
                   b Using ANOVA (type 3) with factors: BIOPOP, BIOPOP. Records with missing values for factors or response were excluded from statistical analyses. 
               
               
                 Frequency distribution of covariates is compared between evaluable and non-evaluable populations 
               
               
                 Note: 
               
               
                 Western Europe = Germany, Italy, Spain, United Kingdom; Eastern Europe = Russian Federation; Other countries = Australia, Korea 
               
             
          
         
       
     
         [0126]    Disease Characteristics at Baseline 
         [0127]    Disease characteristics at baseline were similar in the two populations (see Tables 2 and 3 below). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Summary of disease characteristics at initial diagnosis 
               
             
          
           
               
                   
                 Biomarkers non 
                 Biomarkers 
                   
               
               
                   
                 evaluable population 
                 evaluable population 
               
             
          
           
               
                   
                   
                 Aflibercept/ 
                   
                 Aflibercept/ 
                   
               
               
                   
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
               
               
                   
                 (N = 57) 
                 (N = 49) 
                 (N = 60) 
                 (N = 70) 
                 p-value 
               
               
                   
                   
               
             
          
           
               
                 Primary site [n(%)] 
                   
                   
                   
                   
                 0.7045 a   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
             
          
           
               
                 Colon 
                 27 
                 (47.4%) 
                 26 
                 (53.1%) 
                 31 
                 (51.7%) 
                 33 
                 (47.1%) 
                   
               
               
                 Recto sigmoid 
                 9 
                 (15.8%) 
                 15 
                 (30.6%) 
                 16 
                 (26.7%) 
                 19 
                 (27.1%) 
               
               
                 Rectum 
                 21 
                 (36.8%) 
                 8 
                 (16.3%) 
                 13 
                 (21.7%) 
                 18 
                 (25.7%) 
               
             
          
           
               
                 Histology type [n(%)] 
                   
                   
                   
                   
                   
               
               
                 Number 
                 57 
                 49 
                 60 
                 70 
               
             
          
           
               
                 Adenocarcinoma 
                 57 
                 (100%) 
                 49 
                 (100%) 
                 60 
                 (100%) 
                 70 
                 (100%) 
                   
               
             
          
           
               
                 Staging at diagnosis [n(%)] 
                   
                   
                   
                   
                 0.2297 a   
               
               
                 Number 
                 56 
                 48 
                 57 
                 68 
               
             
          
           
               
                 Stage I 
                 1 
                 (1.8%) 
                  0 
                 2 
                 (3.5%) 
                 2 
                 (2.9%) 
                   
               
             
          
           
               
                 Stage II 
                 2 
                 (3.6%) 
                 1 
                 (2.1%) 
                 6 
                 (10.5%) 
                 4 
                 (5.9%) 
                   
               
               
                 Stage III 
                 5 
                 (8.9%) 
                 3 
                 (6.3%) 
                 3 
                 (5.3%) 
                 4 
                 (5.9%) 
               
               
                 Stage IV 
                 48 
                 (85.7%) 
                 44 
                 (91.7%) 
                 46 
                 (80.7%) 
                 58 
                 (85.3%) 
               
             
          
           
               
                 Time from diagnosis 
                   
                   
                   
                   
                 0.6620 b   
               
               
                 to randomization 
               
               
                 (months)* 
               
               
                 Number 
                 57 
                 49 
                 60 
                 69 
               
               
                 Median 
                   1.4 
                   1.8 
                   1.6 
                   1.7 
               
             
          
           
               
                 Mean (SD) 
                 12.6 
                 (30.9) 
                 9.2 
                 (16.9) 
                 9.8 
                 (19.8) 
                 9.7 
                 (17.5) 
                   
               
             
          
           
               
                 Min:Max 
                 0:149 
                 0:80 
                 0:84 
                 0:80 
               
               
                   
               
               
                   a comparing frequency distribution based on Fisher&#39;s exact test - 2-sided. 
               
               
                   b Using ANOVA (type 3) with factors: BIOPOP, BIOPOP. Records with missing values for factors or response were excluded from statistical analyses. 
               
               
                 Frequency distribution of covariates is compared between evaluable and non-evaluable populations 
               
               
                 *If the day of initial date of diagnosis is missing, it is considered as the first day of the month 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Summary of organs involved at baseline 
               
             
          
           
               
                   
                 Biomarkers non 
                 Biomarkers 
                   
               
               
                   
                 evaluable population 
                 evaluable population 
               
             
          
           
               
                   
                   
                 Aflibercept/ 
                   
                 Aflibercept/ 
                   
               
               
                   
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
               
               
                   
                 (N = 57) 
                 (N = 49) 
                 (N = 60) 
                 (N = 70) 
                 p-value 
               
               
                   
                   
               
             
          
           
               
                 Number of 
                   
                   
                   
                   
                 0.1711 a   
               
               
                 metastatic organs 
               
               
                 involved at baseline 
               
               
                 (excluding primary 
               
               
                 site) [n(%)] 
               
               
                 Number 
                 57 
                 49  
                 60 
                 70 
               
             
          
           
               
                   0 
                  0 
                 0 
                 1 
                 (1.7%) 
                  0 
                   
               
             
          
           
               
                   1 
                 16 
                 (28.1%) 
                 15 
                 (30.6%) 
                 15 
                 (25.0%) 
                 12 
                 (17.1%) 
                   
               
               
                 &gt;1 
                 41 
                 (71.9%) 
                 34 
                 (69.4%) 
                 44 
                 (73.3%) 
                 58 
                 (82.9%) 
               
               
                 Metastatic organs 
                 57 
                 (100%) 
                 49 
                 (100%) 
                 59 
                 (98.3%) 
                 70 
                 (100%) 
               
               
                 involved at baseline 
               
               
                 (excluding primary 
               
               
                 site) [n(%)]* 
               
               
                 Liver 
                 44 
                 (77.2%) 
                 42 
                 (85.7%) 
                 47 
                 (78.3%) 
                 57 
                 (81.4%) 
               
               
                 Lung 
                 27 
                 (47.4%) 
                 18 
                 (36.7%) 
                 25 
                 (41.7%) 
                 28 
                 (40.0%) 
               
               
                 Lymph nodes 
                 26 
                 (45.6%) 
                 25 
                 (51.0%) 
                 30 
                 (50.0%) 
                 38 
                 (54.3%) 
               
               
                 Muscle/soft tissue 
                 9 
                 (15.8%) 
                 6 
                 (12.2%) 
                 14 
                 (23.3%) 
                 10 
                 (14.3%) 
               
               
                 Peritoneum 
                 8 
                 (14.0%) 
                 7 
                 (14.3%) 
                 8 
                 (13.3%) 
                 16 
                 (22.9%) 
               
               
                 Pleura 
                 5 
                 (8.8%) 
                 7 
                 (14.3%) 
                 2 
                 (3.3%) 
                 1 
                 (1.4%) 
               
             
          
           
               
                 Adrenal 
                 2 
                 (3.5%) 
                 0 
                 1 
                 (1.7%) 
                 1 
                 (1.4%) 
                   
               
             
          
           
               
                 Bone 
                 2 
                 (3.5%) 
                 1 
                 (2.0%) 
                 4 
                 (6.7%) 
                 4 
                 (5.7%) 
                   
               
             
          
           
               
                 Kidneys 
                 1 
                 (1.8%) 
                 0 
                  0 
                  0 
                   
               
             
          
           
               
                 Spleen 
                 1 
                 (1.8%) 
                 1 
                 (2.0%) 
                 1 
                 (1.7%) 
                 1 
                 (1.4%) 
                   
               
             
          
           
               
                 Bladder 
                  0 
                 0 
                 1 
                 (1.7%) 
                 1 
                 (1.4%) 
                   
               
             
          
           
               
                 Metastatic organs 
                   
                   
                   
                   
                 0.3536 a   
               
               
                 involved at baseline 
               
               
                 class (excluding 
               
               
                 primary site) [n(%)] 
               
               
                 Number 
                 57 
                 49  
                 60 
                 70 
               
             
          
           
               
                 No liver metastasis, 
                 49 
                 (86.0%) 
                 39 
                 (79.6%) 
                 51 
                 (85.0%) 
                 63 
                 (90.0%) 
                   
               
               
                 or liver and other 
               
               
                 metastases 
               
               
                 Liver metastasis 
                 8 
                 (14.0%) 
                 10 
                 (20.4%) 
                 9 
                 (15.0%) 
                 7 
                 (10.0%) 
               
               
                 only 
               
               
                   
               
               
                   a comparing frequency distribution based on Fisher&#39;s exact test - 2-sided. Records with missing values for factors or response were excluded from statistical analyses. 
               
               
                 Frequency distribution of covariates is compared between evaluable and non-evaluable populations 
               
               
                 *Percentages are not additive (sum greater than 100%) 
               
             
          
         
       
     
         [0128]    Safety Evaluation 
         [0129]    A. Extent of Exposure 
         [0130]    Table 4 below shows that patients in the biomarkers evaluable population were exposed slightly longer to treatment than patients in the biomarkers non-evaluable population (median number of cycles: 12 versus 9 or 10). 
         [0131]    There was no difference in exposure between treatment arms in the biomarkers evaluable population. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Summary of overall study treatment exposure 
               
             
          
           
               
                   
                 Biomarkers non 
                 Biomarkers 
               
               
                   
                 evaluable population 
                 evaluable population 
               
             
          
           
               
                   
                   
                 Aflibercept/ 
                   
                 Aflibercept/ 
               
               
                   
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
                 mFolfox6 
               
               
                   
                 (N = 56) 
                 (N = 49) 
                 (N = 60) 
                 (N = 70) 
               
               
                   
                   
               
             
          
           
               
                 Number of cycles 
                   
                   
                   
                   
               
               
                 received by patient 
               
               
                 Sum 
                 614   
                 572   
                 770.0  
                 865.0  
               
             
          
           
               
                 Mean (SD) 
                 11.0 
                 (7.0) 
                 11.7 
                 (9.5) 
                 12.8 
                 (7.0) 
                 12.4 
                 (7.9) 
               
             
          
           
               
                 Median 
                 10.0 
                  9.0 
                  12.0 
                 12.0 
               
               
                 Min:Max 
                 1:43 
                 1:44  
                 3:33 
                 1:42 
               
               
                 Number of cycles 
               
               
                 received by patient 
               
             
          
           
               
                 1 
                 2 
                 (3.6%) 
                 3 
                 (6.1%) 
                 0 
                 2 
                 (2.9%) 
               
               
                 2 
                 2 
                 (3.6%) 
                 2 
                 (4.1%) 
                 0 
                 2 
                 (2.9%) 
               
             
          
           
               
                 3 
                 3 
                 (5.4%) 
                 1 
                 (2.0%) 
                 2 
                 (3.3%) 
                 2 
                 (2.9%) 
               
               
                 4 
                 3 
                 (5.4%) 
                 4 
                 (8.2%) 
                 5 
                 (8.3%) 
                 8 
                 (11.4%) 
               
             
          
           
               
                 5 
                 1 
                 (1.8%) 
                 0  
                 1 
                 (1.7%) 
                 2 
                 (2.9%) 
               
             
          
           
               
                 6 
                 1 
                 (1.8%) 
                 6 
                 (12.2%) 
                 0 
                 4 
                 (5.7%) 
               
             
          
           
               
                 7 
                 1 
                 (1.8%) 
                 5 
                 (10.2%) 
                 1 
                 (1.7%) 
                 3 
                 (4.3%) 
               
               
                 8 
                 6 
                 (10.7%) 
                 1 
                 (2.0%) 
                 11 
                 (18.3%) 
                 2 
                 (2.9%) 
               
             
          
           
               
                 9 
                 6 
                 (10.7%) 
                 3 
                 (6.1%) 
                 0 
                 5 
                 (7.1%) 
               
             
          
           
               
                 10  
                 5 
                 (8.9%) 
                 2 
                 (4.1%) 
                 4 
                 (6.7%) 
                 3 
                 (4.3%) 
               
               
                 11-15 
                 16 
                 (28.6%) 
                 11 
                 (22.4%) 
                 21 
                 (35.0%) 
                 13 
                 (18.6%) 
               
               
                 16-20 
                 7 
                 (12.5%) 
                 4 
                 (8.2%) 
                 6 
                 (10.0%) 
                 14 
                 (20.0%) 
               
               
                 21-25 
                 1 
                 (1.8%) 
                 3 
                 (6.1%) 
                 5 
                 (8.3%) 
                 6 
                 (8.6%) 
               
               
                 &gt;25    
                 2 
                 (3.6%) 
                 4 
                 (8.2%) 
                 4 
                 (6.7%) 
                 4 
                 (5.7%) 
               
             
          
           
               
                 Duration of 
                   
                   
                   
                   
               
               
                 exposure (weeks) 
               
               
                 Number 
                 56     
                 49     
                 60  
                 70     
               
             
          
           
               
                 Mean (SD) 
                 25.2 
                 (16.0) 
                 27.5 
                 (22.4) 
                 29.5 
                 (16.4) 
                 28.7 
                 (18.7) 
               
             
          
           
               
                 Median 
                 24.1 
                 23.1 
                  27.3 
                 25.4 
               
               
                 Min:Max 
                 2:95 
                 2:106 
                 6:77 
                 2:88 
               
               
                   
               
               
                 Duration of exposure = ((First date of last cycle + 14) − First date of first cycle)/7 
               
               
                 SD: standard deviation 
               
             
          
         
       
     
       B. Plasma Profiling 
       [0132]    The plasma concentration of 27 cytokines, growth factors or soluble receptors was determined by enzyme-linked immunosorbent assays (ELISA) using two Fluorokine® MAP kits (the human angiogenesis panel A and the human high sensitivity cytokine panel; R&amp;D Systems). Competition experiments were conducted to test interference of aflibercept with the detection of VEGF-A, VEGF-D and placental growth factor (PIGF). Angiopoietin-2 (ANGPT2), SDF1-α, HGF, VEGF-C, soluble VEGF receptor 3 (sFLT4, sVEGFR3) and sVEGFR2 were assessed by single ELISA (R&amp;D Systems). Plasma markers were analyzed at baseline, at 30 and 60 days after the first study treatment infusion and 30 days after the last aflibercept infusion. 
       Statistical Analysis 
       [0133]    Differences between patients with evaluable biomarkers and patients without evaluable biomarkers were assessed using a two-sided Fisher&#39;s exact test for categorical variables and ANOVA for continuous variables. Biomarkers were analyzed as quantitative variables, by coding the absence or presence of a somatic mutation as 0 or 1, and SNP genotypes as 0, 1 or 2 depending on the number of minor alleles present. The linear effects of baseline biomarkers on PFS were assessed using a Cox proportional hazard model with the following co-variates: Eastern Cooperative Oncology Group (ECOG) performance status (0-1 versus 2), liver-only metastases (yes/no), and the number of distant metastasis organs (1 versus &gt;1), a treatment effect, a biomarker effect and a biomarker-treatment interaction effect. The significance of the latter two effects was jointly tested by a two-degrees-of-freedom Wald test. Extended statistical methods are described in supplementary methods. 
       Results 
       [0134]    Of the 236 patients in the ITT population of the AFFIRM trial 227 (96%) were evaluable for response. Of these, 130 (57%) provided at least one biological sample, 60 (46%) and 70 (54%) of which participated in the mFOLFOX6 and mFOLFOX6 plus aflibercept arms, respectively. There was no major difference at a false discovery rate (FDR)-adjusted P-value of 0.05 between patients who provided a biological sample and those who did not in terms of patient biometrics, ethnicity, and disease characteristics at baseline, or at efficacy and safety endpoints (Table 1). Of those who provided at least one biological sample, 51 (39%) provided samples for each of the 3 biomarker types, with 88 (68%) and 97 (74%) patients providing samples for 2 or 1 of the biomarker types respectively. Each biomarker type was analyzed separately, to avoid patient groups that were too small for sub-analyses. 
       Profiling of Plasma Markers for Efficacy 
       [0135]    Plasma levels of 27 markers were measured at different time points (i.e., at baseline [87 patients]; 30 and 60 days after start of treatment [82 and 73 patients]; and 30 days after the last treatment [56 patients] as indicated on Table 5. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Number of observations per time point - total, below limit of quantification 
               
               
                 (LOQ) and of detection (LOD) 
               
             
          
           
               
                   
                   
                   
                   
                 EOT + 30 
               
               
                   
                 Baseline 
                 Day 30 
                 Day 60 
                 Days 
               
             
          
           
               
                   
                 Total 
                 &lt;LOQ 
                 &lt;LOD 
                 Total 
                 &lt;LOQ 
                 &lt;LOD 
                 Total 
                 &lt;LOQ 
                 &lt;LOD 
                 Total 
                 &lt;LOQ 
                 &lt;LOD 
               
               
                   
                   
               
             
          
           
               
                 ANGPT1 
                 87 
                 1 
                 1 
                 80 
                 1 
                 0 
                 73 
                 0 
                 0 
                 56 
                 0 
                 0 
               
               
                 ANGPT2 
                 86 
                 1 
                   
                 82 
                 1 
                   
                 72 
                 0 
                   
                 55 
                 1 
               
               
                 CSF2 
                 84 
                 28 
                 17 
                 80 
                 38 
                 16 
                 72 
                 32 
                 17 
                 54 
                 25 
                 16 
               
               
                 CXCL12 
                 86 
                 0 
                 0 
                 82 
                 2 
                 2 
                 72 
                 0 
                 0 
                 55 
                 0 
                 0 
               
               
                 FGF1 
                 87 
                 14 
                 14 
                 80 
                 16 
                 16 
                 73 
                 18 
                 18 
                 56 
                 8 
                 8 
               
               
                 Endostatin 
                 87 
                   
                   
                 80 
                   
                   
                 73 
                   
                   
                 56 
               
               
                 FGF2 
                 87 
                 19 
                 11 
                 80 
                 20 
                 15 
                 73 
                 19 
                 11 
                 56 
                 10 
                 5 
               
               
                 FIGF 
                 87 
                 12 
                 12 
                 80 
                 4 
                 4 
                 73 
                 1 
                 1 
                 56 
                 1 
                 1 
               
               
                 HGF 
                 86 
                 0 
                 0 
                 82 
                 7 
                 7 
                 72 
                 1 
                 1 
                 55 
                 1 
                 1 
               
               
                 IFNG 
                 84 
                 6 
                 4 
                 80 
                 8 
                 3 
                 72 
                 6 
                 2 
                 54 
                 11 
                 8 
               
               
                 IL10 
                 84 
                 0 
                 0 
                 80 
                 1 
                 1 
                 72 
                 2 
                 2 
                 54 
                 0 
                 0 
               
               
                 IL12 
                 84 
                 3 
                 3 
                 80 
                 1 
                 1 
                 72 
                 3 
                 3 
                 54 
                 8 
                 7 
               
               
                 IL1B 
                 84 
                 1 
                 1 
                 80 
                 0 
                 0 
                 72 
                 1 
                 1 
                 54 
                 4 
                 4 
               
               
                 IL2 
                 84 
                 3 
                 2 
                 80 
                 3 
                 1 
                 72 
                 1 
                 1 
                 54 
                 8 
                 6 
               
               
                 IL4 
                 84 
                 2 
                 2 
                 80 
                 1 
                 1 
                 72 
                 2 
                 2 
                 54 
                 7 
                 7 
               
               
                 IL5 
                 84 
                 2 
                 1 
                 80 
                 3 
                 0 
                 72 
                 1 
                 0 
                 54 
                 1 
                 1 
               
               
                 IL6 
                 84 
                 0 
                 0 
                 80 
                 2 
                 1 
                 72 
                 0 
                 0 
                 54 
                 1 
                 1 
               
               
                 IL8 
                 84 
                 0 
                 0 
                 80 
                 1 
                 1 
                 72 
                 0 
                 0 
                 54 
                 0 
                 0 
               
               
                 PGF 
                 87 
                 8 
                 8 
                 80 
                 6 
                 6 
                 73 
                 2 
                 2 
                 56 
                 1 
                 1 
               
               
                 TNF 
                 84 
                 2 
                 1 
                 80 
                 2 
                 1 
                 72 
                 1 
                 0 
                 54 
                 2 
                 1 
               
               
                 PDGFA 
                 87 
                   
                   
                 80 
                   
                   
                 73 
                   
                   
                 56 
               
               
                 VEGFA 
                 84 
                 6 
                 1 
                 80 
                 4 
                 1 
                 72 
                 2 
                 0 
                 54 
                 4 
                 0 
               
               
                 PDGFB 
                 87 
                   
                   
                 80 
                   
                   
                 73 
                   
                   
                 56 
               
               
                 VEGFC 
                 86 
                 1 
                 1 
                 82 
                 0 
                 0 
                 72 
                 0 
                 0 
                 55 
                 1 
                 0 
               
               
                 sFLT4 
                 86 
                 0 
                 0 
                 82 
                 7 
                 7 
                 72 
                 1 
                 1 
                 55 
                 1 
                 1 
               
               
                 THBS2 
                 87 
                   
                   
                 80 
                   
                   
                 73 
                   
                   
                 56 
               
               
                 sKDR 
                 86 
                 0 
                 0 
                 82 
                 7 
                 7 
                 72 
                 1 
                 1 
                 55 
                 1 
                 1 
               
               
                   
               
             
          
         
       
     
         [0136]    All cytokines were measured in pg/ml, but some transformations were applied when necessary to obtain a symmetric distribution or smaller numerical values (ng/ml) for the association models, as depicted in table 6. 
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 Selected transformations of original plasma cytokine levels (pg/ml) 
               
             
          
           
               
                   
                 Cytokine 
                 Transformation 
               
               
                   
                   
               
               
                   
                 ANGPT1 
                 log 
               
               
                   
                 ANGPT2 
                 log 
               
               
                   
                 CSF2 
                 square root 
               
               
                   
                 CXCL12 
                 ng/ml 
               
               
                   
                 FGF1 
                 cubic root 
               
               
                   
                 Endostatin 
                 log 
               
               
                   
                 FGF2 
                 square root 
               
               
                   
                 FIGF 
                 cubic root 
               
               
                   
                 HGF 
                 Log 
               
               
                   
                 IFNG 
                 square root 
               
               
                   
                 IL10 
                 cubic root 
               
               
                   
                 IL12 
                 None 
               
               
                   
                 IL1B 
                 cubic root 
               
               
                   
                 IL2 
                 square root 
               
               
                   
                 IL4 
                 None 
               
               
                   
                 IL5 
                 Log 
               
               
                   
                 IL6 
                 Log 
               
               
                   
                 IL8 
                 Log 
               
               
                   
                 PGF 
                 ng/ml 
               
               
                   
                 TNF 
                 square root 
               
               
                   
                 PDGFA 
                 log 
               
               
                   
                 VEGFA 
                 Log 
               
               
                   
                 PDGFB 
                 log 
               
               
                   
                 VEGFC 
                 Log 
               
               
                   
                 sFLT4 
                 square root(ng/ml) 
               
               
                   
                 THBS2 
                 log 
               
               
                   
                 sKDR 
                 square root(ng/ml) 
               
               
                   
                   
               
             
          
         
       
     
         [0137]    We assessed the association of each plasma marker at baseline with PFS, while allowing for an interaction with treatment (Table 7). The lowest P-value was obtained for IL8 (P=0.0211; FDR=0.596 and P=0.0218 for interaction). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Effect of baseline plasma biomarkers on PFS. The P- 
               
               
                 values associated to the joint effect, the FDR-corrected 
               
               
                 joint effect, the plasma biomarker and the biomarker 
               
               
                 with treatment interaction effect are shown. 
               
             
          
           
               
                   
                 P-values 
                   
               
             
          
           
               
                   
                   
                   
                 FDR- 
                   
                 Treatment 
               
               
                   
                   
                   
                 corrected 
                 Plasma 
                 by plasma 
               
               
                   
                   
                 Joint 
                 joint 
                 level 
                 level 
               
               
                   
                 Protein 
                 effect 
                 effect 
                 effect 
                 effect 
               
               
                   
                   
               
               
                   
                 IL8 
                 0.0221 
                 0.5962 
                 0.6701 
                 0.0218 
               
               
                   
                 THBS2 
                 0.1408 
                 0.6523 
                 0.0545 
                 0.2234 
               
               
                   
                 CXCL12 
                 0.1610 
                 0.6523 
                 0.0573 
                 0.1640 
               
               
                   
                 IL10 
                 0.1669 
                 0.6523 
                 0.0647 
                 0.1311 
               
               
                   
                 Ang1 
                 0.1768 
                 0.6523 
                 0.0724 
                 0.0831 
               
               
                   
                 FIGF 
                 0.1974 
                 0.6523 
                 0.6826 
                 0.1704 
               
               
                   
                 FGF2 
                 0.2063 
                 0.6523 
                 0.1722 
                 0.0768 
               
               
                   
                 sVEGFR2 
                 0.2171 
                 0.6523 
                 0.1616 
                 0.0812 
               
               
                   
                 PDGFA 
                 0.2474 
                 0.6523 
                 0.1155 
                 0.1052 
               
               
                   
                 IL6 
                 0.2689 
                 0.6523 
                 0.1061 
                 0.1838 
               
               
                   
                 FGF1 
                 0.3185 
                 0.6523 
                 0.4759 
                 0.1536 
               
               
                   
                 VEGF-A 
                 0.3202 
                 0.6523 
                 0.2549 
                 0.7133 
               
               
                   
                 CSF2 
                 0.3272 
                 0.6523 
                 0.4278 
                 0.1679 
               
               
                   
                 IL12 
                 0.3587 
                 0.6523 
                 0.3664 
                 0.1808 
               
               
                   
                 IFNg 
                 0.3855 
                 0.6523 
                 0.8662 
                 0.2984 
               
               
                   
                 IL4 
                 0.3866 
                 0.6523 
                 0.7326 
                 0.4796 
               
               
                   
                 PIGF 
                 0.5008 
                 0.7955 
                 0.3018 
                 0.2595 
               
               
                   
                 PDGFB 
                 0.5442 
                 0.8163 
                 0.4795 
                 0.2963 
               
               
                   
                 IL1B 
                 0.6025 
                 0.8439 
                 0.3183 
                 0.3595 
               
               
                   
                 HGF 
                 0.6286 
                 0.8439 
                 0.3369 
                 0.5093 
               
               
                   
                 IL2 
                 0.6564 
                 0.8439 
                 0.7696 
                 0.4093 
               
               
                   
                 Endostatin 
                 0.9021 
                 0.9783 
                 0.7429 
                 0.9824 
               
               
                   
                 sFLT4 
                 0.9063 
                 0.9783 
                 0.6849 
                 0.8668 
               
               
                   
                 TNF 
                 0.9461 
                 0.9783 
                 0.8736 
                 0.7442 
               
               
                   
                 Ang2 
                 0.9698 
                 0.9783 
                 0.9279 
                 0.9747 
               
               
                   
                 VEGF-C 
                 0.9772 
                 0.9783 
                 0.8375 
                 0.8307 
               
               
                   
                 IL5 
                 0.9783 
                 0.9783 
                 0.8813 
                 0.8501 
               
               
                   
                   
               
             
          
         
       
     
         [0138]    The Cox model assumes a linear relationship between the plasma marker and the log of the PFS hazard function, but since this hypothesis may be violated when analyzing continuous markers that vary considerably, a threshold effect may be more relevant. We explored this possibility by searching for the optimal cut-off level that maximizes the interaction with the treatment and the plasma marker. For IL8, the optimal cut-off was at 19 pg/ml (77 th  percentile). A model, in which IL8 was analyzed as a binary variable with this threshold, fitted better than a model with continuous IL8 levels (AIC of 469.3 versus 477.6). Patients with low IL8 levels 9 pg/ml, 77% of patients) exhibited a longer PFS in the aflibercept/mFLOFOX6 arm than in the mFOLFOX6 arm (Table 8). 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 Kaplan-Meier estimates of effect of biomarkers on months of PFS. 
               
             
          
           
               
                   
                   
                 Aflibercept plus 
                 Hazard Ratio 
               
               
                 Plasma protein 
                 mFOLFOX6 Median 
                 mFOLFOX6 Median 
                 vs mFOLFOX6 
               
               
                 markers 
                 (99% CI) 
                 (99% CI) 
                 (99% CI) 
               
               
                   
               
             
          
           
               
                 All patients 
                 8.8 (6.57-10.02) 
                 8.5 
                 (6.67-10.05) 
                 0.979 
                 (0.505-1.897) 
               
               
                 IL8 ≦ 19 pg/mL 
                 8.8 (5.62-10.91) 
                 9.3 
                 (7.52-11.10) 
                 0.764 
                 (0.363-1.607) 
               
               
                 IL8 &gt; 19 pg/mL 
                 8.8 (5.09-15.64) 
                 4.1 
                 (2.33-8.54) 
                 2.71 
                 (0.735-9.984) 
               
               
                   
               
               
                 CI, confidence interval; mt, mutant; wt, wild-type. 
               
             
          
         
       
     
         [0139]    We also analyzed whether treatment-related changes in plasma markers could predict aflibercept treatment outcome. The Cox model included the effect of baseline plasma levels and the difference in expression measured at baseline and the last time point before discontinuation, disease progression or death (i.e., at 30 days or 60 days of treatment), while allowing for interaction with the treatment arm. IL8 was the only marker with a significant effect of change from baseline on PFS (P=0.0018; FDR=0.0478; Table 9). This effect did not differ between treatment arms (P=0.2028). High baseline or post-baseline increased IL8 levels corresponded to a higher probability of disease progression at 12 months ( FIGS. 1 and 2 ). 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 Effect of plasma marker changes from baseline on PFS. P-values 
               
               
                 (and FDR) of the joint effects of plasma marker and treatment 
               
               
                 by plasma marker interactions are shown. P-values for the change 
               
               
                 from baseline and the interaction are also presented. 
               
             
          
           
               
                   
                 P-values 
                   
               
             
          
           
               
                   
                   
                   
                 Joint 
                   
                 Treatment 
               
               
                   
                   
                   
                 effect 
                 Change 
                 by Change 
               
               
                   
                   
                 Joint 
                 FDR- 
                 from base- 
                 from base- 
               
               
                   
                 Protein 
                 effect 
                 corrected 
                 line effect 
                 line effect 
               
               
                   
                   
               
               
                   
                 IL8 
                 0.0018 
                 0.0478 
                 0.0006 
                 0.2028 
               
               
                   
                 IL10 
                 0.0342 
                 0.4525 
                 0.5214 
                 0.8204 
               
               
                   
                 VEGFA 
                 0.0619 
                 0.4525 
                 0.0189 
                 0.0704 
               
               
                   
                 CXCL12 
                 0.0670 
                 0.4525 
                 0.1714 
                 0.0318 
               
               
                   
                 CSF2 
                 0.0855 
                 0.4619 
                 0.0266 
                 0.1682 
               
               
                   
                 VEGFC 
                 0.1127 
                 0.5072 
                 0.0742 
                 0.9355 
               
               
                   
                 IL5 
                 0.1886 
                 0.7275 
                 0.0684 
                 0.1657 
               
               
                   
                 Endostatin 
                 0.2418 
                 0.7882 
                 0.9988 
                 0.2668 
               
               
                   
                 PDGFA 
                 0.3092 
                 0.7882 
                 0.1508 
                 0.9922 
               
               
                   
                 TNF 
                 0.3209 
                 0.7882 
                 0.1770 
                 0.1554 
               
               
                   
                 IL4 
                 0.3211 
                 0.7882 
                 0.1938 
                 0.7631 
               
               
                   
                 FGF2 
                 0.3851 
                 0.8191 
                 0.6468 
                 0.3957 
               
               
                   
                 sFLT4 
                 0.3944 
                 0.8191 
                 0.2533 
                 0.1729 
               
               
                   
                 THBS2 
                 0.4677 
                 0.8426 
                 0.2387 
                 0.6683 
               
               
                   
                 FGF1 
                 0.4770 
                 0.8426 
                 0.4669 
                 0.2242 
               
               
                   
                 PGF 
                 0.4993 
                 0.8426 
                 0.2501 
                 0.2755 
               
               
                   
                 ANGPT2 
                 0.5638 
                 0.8924 
                 0.3373 
                 0.8394 
               
               
                   
                 IL1B 
                 0.6261 
                 0.8924 
                 0.3345 
                 0.5414 
               
               
                   
                 PDGFB 
                 0.6583 
                 0.8924 
                 0.3730 
                 0.6009 
               
               
                   
                 IL12 
                 0.6611 
                 0.8924 
                 0.7934 
                 0.7744 
               
               
                   
                 IL2 
                 0.7984 
                 0.9482 
                 0.9352 
                 0.6027 
               
               
                   
                 sKDR 
                 0.8375 
                 0.9482 
                 0.6009 
                 0.5613 
               
               
                   
                 HGF 
                 0.8383 
                 0.9482 
                 0.5804 
                 0.5665 
               
               
                   
                 IFNG 
                 0.8559 
                 0.9482 
                 0.6923 
                 0.9101 
               
               
                   
                 ANGPT1 
                 0.8814 
                 0.9482 
                 0.6269 
                 0.8538 
               
               
                   
                 FIGF 
                 0.9131 
                 0.9482 
                 0.6838 
                 0.7528 
               
               
                   
                 IL6 
                 0.9963 
                 0.9963 
                 0.9893 
                 0.9594 
               
               
                   
                   
               
             
          
         
       
     
         [0140]    When plasma biomarkers were measured at baseline only, IL8 had the most prominent effect on PFS, which was best described as a threshold effect with high circulating IL8 (IL8&gt;19 pg/mL) associated with a shorter PFS in the aflibercept-treated patients. 
         [0141]    When plasma biomarkers were measured at baseline and during treatment, high levels of circulating IL8 at baseline together with increased levels of IL8 measured during treatment were significantly associated with reduced PFS (FDR=0.0478). 
         [0142]    Conclusions 
         [0143]    We identified that high IL8 levels at baseline correlated with shorter survival times, and patients with increasing levels of IL8 during treatment were more likely to progress. 
         [0144]    This suggests that patients with high IL8 levels, at baseline or during treatment, are at increased risk of disease progression during aflibercept therapy.