WO2013053918A1

WO2013053918A1 - Diagnosis of intraocular lymphoma

Info

Publication number: WO2013053918A1
Application number: PCT/EP2012/070325
Authority: WO
Inventors: Sylvain Fisson
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2011-10-14
Filing date: 2012-10-12
Publication date: 2013-04-18
Anticipated expiration: 2014-04-14

Abstract

The present invention provides methods and kits for diagnosing primary central nervous system lymphoma, in particular for discriminating patients suffering from primary intraocular lymphoma from patients suffering from uveitis or other related ocular disorders.

Description

DIAGNOSIS OF INTRAOCULAR LYMPHOMA

FIELD OF THE INVENTION The present invention provides methods and kits for diagnosing primary central nervous system lymphoma, in particular for discriminating patients suffering from primary intraocular lymphoma from patients suffering from uveitis or other related ocular disorders. BACKGROUND OF THE INVENTION

Primary intraocular lymphoma (PIOL) is a subset of primary central nervous system lymphoma (PCNSL) that initially presents in the eye with or without simultaneous CNS involvement. The majority of cases of PIOL are related to high grade extranodal non- Hodgkin, diffuse large B-cell lymphomas. PIOL often masquerades as chronic uveitis. Thus, clinical diagnosis of PIOL remains difficult and requires a high degree of suspicion. A high level of IL10 within pure vitreous or aqueous humor samples of PIOL or an IL- 10/IL-6 ratio greater than 1 for diluted or undiluted PIOL samples are considered as an indirect diagnostic evidence motivating further investigations (Cassoux et al., 2007; Chan et al., 2009). Indeed, the exact cutoff ratio indicating a positive test result may vary between laboratories and techniques used. Moreover, false positive (11%) or negative (23 to 30%) PIOL patients were described (Whitcup et al., 1997; Chan et al., 2003; Wolf et al., 2003; Cassoux et al., 2007). Thus, without a systematic cytological analysis these strategies failed to discriminate some PIOL from uveitis patients (Buggage et al., 1999). A better characterization of the molecular microenvironment is thus crucial in order to find new cytokine combinations as diagnostic markers.

SUMMARY OF THE INVENTION The present invention relates to an in vitro method of diagnosis of primary central nervous system lymphoma, said method comprising:

a) providing a biological sample from a subject,

b) determining the concentrations of at least IL10 and IFNy biomarkers in said biological sample,

wherein said subject is predicted to suffer from primary central nervous system lymphoma when the ratio of concentrations of the biomarkers in said biological sample is such that:

Log([IL10]/[IFNy]) > 0. The method is particularly useful for diagnosing primary intraocular lymphoma (PIOL) from intraocular fluid samples such as vitreous samples. The invention is partly based on unexpected results obtained when determining the levels of certain biomarkers from vitreous samples of patients with PIOL.

The diagnosis method is highly sensitive and carried out from a small volume of sample, without the need of skilled practitioner.

The diagnosis method according to the invention may be conveniently performed using immunoassays, including ELISA, microarray or bead flow cytometry technology.

The invention further relates to the kit for carrying out such diagnosis method, said kit comprising:

o a detectable binding reagent specific of IL10 biomarker,

o a detectable binding reagent specific of IFNy biomarker, and,

o optionally, labelling reagent for detecting said binding reagents and/or appropriate buffers.

In one preferred embodiment, the kit according to the invention, comprises

o A first set of detectable binding reagents comprising:

• an antibody directed against IL10 biomarker,

• an antibody directed against IFNy biomarker, and,

· an antibody directed against IL6 biomarker,

wherein each antibody of said first set is conjugated to fluorescent beads, and the fluorescent beads have different wave-lengths depending upon the biomarker specificity of the conjugated antibody,

o A second set of detectable binding reagents comprising:

· an antibody directed against IL10 biomarker,

• an antibody directed against IFNy biomarker, and,

• an antibody directed against IL6 biomarker,

wherein each antibody of said second set is conjugated to a fluorochrome. Other biomarkers associated to PCNSL are further disclosed in the present invention that can be used alone or in combination with the above identified biomarkers IL10, IFNy and IL6 in the diagnosis methods of the invention. Those biomarkers are selected from the group consisting of sCD25 (soluble CD25), ILIRa, IL-13, IL-16, MIF, MIP-la, Serpin El.

The invention also relates to a method for treating primary central nervous system lymphoma in a patient in need thereof, said method comprising the steps of:

a) selecting the patient in need of a treatment for primary central nervous system lymphoma by carrying out the diagnosis methods according to the invention, b) administering to said patient a therapeutically effective amount of an appropriate drug for treating said primary central nervous system lymphoma.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have shown that combination of novel biomarkers can be used to diagnose PCNSL, in particular PIOL from vitreous samples. In particular the combination of [IL10]/[IL6] and [IL10]/[IFNy] concentration ratios have proved to be useful to discriminate in a first step PIOL-masquerade syndrome among uveitis patients.

Therefore, the present invention provides novel biomarkers that can be used to diagnose PCNSL in a subject.

In one aspect, the present invention relates to an in vitro method of diagnosis of primary central nervous system lymphoma, said method comprising:

a) providing a biological sample from a subject,

Log([IL10]/[IFNy]) > 0.

As used herein, the term "marker" or "biomarker" refers to a molecule (typically a protein, nucleic acid, carbohydrate or lipid) that is present in the biological sample, for example IL10, IFNy or IL6 cytokine, which may have detectable variable forms in vivo or quantifiable variable concentration in said biological sample.

As used herein, the term "diagnosis" refers to the clinical evaluation of the presence or properties of a pathological state. An "in vitro" diagnosis means that the diagnosis method is performed on a biological sample, previously collected from the subject.

With respect to the objects of the present invention, the diagnosis consists of evaluating the absence or presence of primary central nervous system lymphoma, for example, PIOL, in a subject, for example, in a subject presenting some symptoms of uveitis or other related eye disorders. More specifically, the diagnosis methods according to the invention may refer to the discrimination between at least two pathologies with similar symptoms.

As exemplified herein below, specific ratios of concentrations of identified biomarkers correlated with high sensitivity with the clinical diagnosis of primary intraocular lymphoma patients compared to uveitis patients. In particular, [IL10]/[IL6] and/or [IL10]/[IFNy] concentration ratios in vitreous samples may be used in the diagnosis methods of the invention.

In one specific embodiment, IL10 refers to the human interleukin having the amino acid sequence as shown in accession number P22301 (Uniprot database) or its natural variants, including the mature (processed) form of the protein.

In one specific embodiment, IL6 refers to the human interleukin having the amino acid sequence as shown in accession number P05231 (Uniprot database) or its natural variants, including the mature (processed) form of the protein.

In one specific embodiment, IFNy refers to the human cytokine having the amino acid sequence as shown in accession number P01579 (Uniprot database) or its natural variants, including the mature (processed) form of the protein.

In one embodiments, the "natural variants" refers to a polypeptide with at least 70%, at least 75%, at least 80%, at least 85%, at least 90% identity to the biomarker's reference amino acid sequence listed above, which is detected in said biological sample instead of the wild type corresponding biomarker, the amino acid variations resulting from natural mutations in the corresponding gene or mRNA.

As used herein, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i. e., % identity = # of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below.

The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

As used herein, the term "subject" refers to a mammal, for example human, primates, horses, cats, dogs, mice, preferably, a human, and preferably a human patient presenting one or more symptoms of a patient suffering from inflammatory ocular disorders, such as uveitis.

The term "biological sample" includes fresh and frozen tissues such as biopsy or autopsy samples. Biological samples may include ocular fluids, blood and blood fractions or other body fluids, including cerebrospinal fluid, lymph, plasma, urine, sputum, or other secretions. In one preferred embodiment, in particular for diagnosing primary intraocular lymphoma, the term "biological sample" includes samples from intraocular fluids, including vitreous and aqueous humors. Intraocular fluids or tissue sections may be obtained by vitrectomy or vitreal or fine needle aspiration.

In one specific embodiment, the method is carried out on a vitreous sample (diluted or undiluted) of a volume that does not exceed ΙΟΟμΙ, for example a volume of 50μ1 or less.

The method according to the invention comprises determining the concentrations of at least IL10 and IFNy biomarkers in said biological sample and/or the corresponding concentration ratios [IL10]/[IFNy].

In one preferred embodiments, the concentration of IL6 is further determined, and/or the corresponding concentration ratio [IL10]/[IL6] in combination with [IL10]/[IFNy]. When both ratios are superior or equal to 1 (or their Log is superior or equal to 0), this indicates that the subject is suffering from primary central nervous system lymphoma, for example, primary intraocular lymphoma.

Since the diagnosis method is based on the ratio of concentrations of biomarkers, it is not necessary, according to a specific embodiment of the method, to determine the absolute concentration of each biomarker. Determining their relative concentration from one to another may be sufficient. Thus, advantageously, the biological sample may be either non diluted or diluted vitreous samples, and the order of dilution of the biological sample may be unkown.

The concentrations of the biomarkers, for example relative vitreal concentrations of IL10, IL6 and/or IFNy may be determined according to any appropriate detection techniques.

In one embodiment, antibody reagents can be used to determine levels of biomarkers in the biological sample, using any of the number of immunoassays known to those skilled in the Art. The term "immunoassay" encompasses techniques including without limitation, enzyme immunoassays (EIA) such as enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), microparticle enzyme immunoassay (MEIA), capillary electrophoresis immunoassays (CEIA), radioimmunoassay (RIA), immunoradiometric assays (IRMA), fluorescence polarization immunoassays (FPIA) and chemiluminescence assays (CL). Specific immunological binding of the antibody to the biomarker can be detected directly or indirectly.

As used herein, the term "antibody" includes whole antibodies and any antigen binding fragments (i.e., "antigen-binding portion") or single chains thereof.

A naturally occurring "antibody" is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V_H) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V_L) and a light chain constant region. The light chain constant region is comprised of one domain, C_L- The V_H and V_L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V_H and V_L is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antigen-binding portion" of an antibody (or simply "antigen portion"), as used herein, refers to full length or one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a portion of IL10, IFNy or IL6 biomarker). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the V_L, V_H, C_L and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR), or any fusion proteins comprising such antigen-binding portion.

Furthermore, although the two domains of the Fv fragment, V_L and V_H, are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single chain protein in which the V_L and V_H regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al, 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen- binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

As used herein a "specific" binding refers to a reagent (for example an antibody) that is able to bind to the biomarker with a KD of at least ΙΟΟμΜ or less, ΙΟμΜ or less, ΙμΜ or less, lOOnM or less, or lOnM or less. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of K_d to K_a (i.e. K_d/K_a) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A method for determining the KD of an antibody is by using surface plasmon resonance, or using a biosensor system such as a Biacore^® system.

Antibodies for detecting the biomarkers of the invention may be purified or synthesized using methods well known in the art, e.g. by immunization with a cytokine identified as a biomarker in the present invention, or recombinantly produced or purchased when commercially available. Antibodies against IL6, IL10 or IFNy are for example commercially available from BD Biosciences, Bender, Diaclone or Luminex.

Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.

A signal from direct or indirect labeling can be then analysed, for example, using a spectrophotometer to detect color from a chromogenic substrate, a radiation counter to detect radiation, or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.

The antibody reagents for use in the methods of the invention to detect the biomarkers, can be immobilized onto a variety of solid supports, such as polystyrene beads, magnetic or chromatographic matrix particles, the surface of an assay plate, glass or nylon supports.

In one preferred embodiment, the biomarkers concentrations are determined using multiplex fluorescent bead-based technology, such as cytometric bead array technology. Cytometric Bead Array technology (CBA kit, BD Bioscience) is a multiplex technology comprising

(i) a fist set of capture bead populations with distinct fluorescent dyes, coated with cytokine- specific capture antibodies; (ii) a second set of labeled cytokine- specific capture antibodies, for example labeled with a flurochrome such as phycoerythrine.

Both capture bead populations and capture antibodies are mixed together in appropriate proportion with the biological sample to form sandwich complexes. The beads are then washed and the data are acquired using flow cytometer. By selecting distinct fluorescence wave-lengths depending upon the specificity of the coated antibodies, it is possible to measure in parallel the relative amount of each type bead after binding with the corresponding cytokine and to derive from these data, the relative amount of different cytokines in the sample of a volume lower than ΙΟΟμΙ.

Once the (relative) concentrations of the biomarkers have been determined, a subject is predicted as suffering from a primary central nervous system lymphoma, for example, primary intraocular lymphoma, when the ratio of concentrations of the biomarkers in said biological sample is such that:

Log([IL10]/[IFNy])>0 or Log([IL10]/[IL6])>0

In certain embodiments, the prediction is expressed as a statistical value, including a P value, as calculated from the ratio values obtained from [IL10], [IFNy] and [IL6] concentrations and, optionally, from relative concentrations of one or more other biological markers present in the biological sample.

The diagnosis as such may not necessarily be based only on the above-mentioned cutoff ratios. The final diagnosis may further include other data, including clinical evaluation of the symptoms of the patients, or be based on the combination of other data, such as other cytokine concentrations or cytokine concentrations ratios.

The invention also relates to a kit for carrying out the above diagnosis methods.

In particular, the invention provides a kit for carrying out the above diagnosis methods, comprising:

a. a detectable binding reagent specific of IL10 biomarker,

b. a detectable binding reagent specific of IFNy biomarker,

c. optionally labeling reagent for detecting said binding reagents and/or appropriate buffers.

The same kit may further comprise binding reagent specific of IL6 biomarker, or other biomarkers selected from the group consisting of: sCD25 (soluble CD25), ILIRa, IL-13, IL-16, MIF, MIP-la, Serpin El.

The "binding reagent" of the kits according to the invention may be any binding compound that is able to specifically bind to the biomarker. Binding reagent may be antibody or non- antibody scaffold. Non antibody scaffold includes, without limitation, Adnectins (fibronectin) (Adnexus, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AG, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

In one preferred embodiment, a detectable binding reagent is an antibody conjugated to a solid support, for example conjugated to a bead.

In certain embodiments, the kit is appropriate for an immunoassay, such as ELISA. In such assays, samples are typically incubated in the presence of an immobilized first specific binding reagent (e.g an antibody against IL10 or IFNy) and binding of the biomarker to said first specific binding reagent using a detectable (for example labeled) second specific binding reagent capable of specifically binding to the biomarker (at a different epitope of the first specific binding reagent).

In other embodiments, the kit is appropriate for flow cytometry assays such as multiplex bead-based assay, for example cytometric bead array (CBA) technology. In such embodiment, the kit may comprise: a. a first set of detectable binding reagents comprising:

i. an antibody directed against IL10 biomarker,

ii. an antibody directed against IFNy biomarker, and, iii. an antibody directed against IL6 biomarker,

wherein each antibody of said first set is conjugated to fluorescent beads, and the fluorescent beads have different wave-length depending upon the biomarker specificity of the conjugated antibody,

b. a second set of detectable binding reagents comprising:

i. an antibody directed against IL10 biomarker,

wherein each antibody of said second set is conjugated to a fluorochrome.

For the first set of detectable binding reagents, advantageously, the beads (i) coated with anti-ILlO antibody are labelled with a fluorescent dye (i), the beads (ii) coated with anti- IFNy antibody are labelled with a fluorescent dye (ii) and the beads (iii) coated with anti- IL6 biomarker are labelled a fluorescent dye (iii), wherein fluorescent dyes (i), (ii) and (iii) have distinct fluorescent properties, so that they can be detected in parallel by multiplex technologies. Alternatively, biomarkers may be captured on antibody microarray. The antibody micro- array comprises anti-biomarker antibodies (for example antibodies specific of IL6, IL10, IFNy and/or other biomarkers selected from the group consisting of: sCD25 (soluble CD25), ILIRa, IL-13, IL-16, MIF, MIP-la, Serpin El.

The kits of the invention may optionally comprise additional components useful for performing the methods of the invention. By way of example, the kits may comprise fluids (e.g. SSC buffer) suitable for binding an antibody with a protein with which it specifically binds, one or more sample compartments, an instructional material which describes performance of the diagnosis method, and the like.

More generally, the biomarkers of the invention may also be used for patient stratification methods, for selecting patients in need of a treatment against PCNSL or more preferably against PIOL and/or for discriminating them from the patients in need of a treatment against uveitis.

In one specific embodiment, the invention relates to a method for treating primary central nervous system lymphoma, in a patient in need thereof, said method comprising the steps of

a) selecting the patient in need of a treatment for primary central nervous system lymphoma by carrying out the diagnosis methods as above defined. b) administering to said patient a therapeutically effective amount of an appropriate treatment or drug for treating said primary central nervous system lymphoma.

In one preferred embodiment, said primary central nervous system lymphoma is a primary intraocular lymphoma. At step b), an appropriate drug for treating PIOL includes, without limitation, anti-CD20 antibody, methotrexate, cytarabine, ifosfamide and trofosfamide and their pharmaceutically acceptable salts and esters. Other appropriate treatments for PIOL include without limitation ocular radiotherapy. Anti-CD20 antibody which may be used in the above treatment methods, includes, without limitation, rituximab, ofatumumab, tozitumomab, ocrelizumab, LFB-R603 and their generic versions.

The above diagnostic method may also be used to monitor the efficacy of a treatment for primary central nervous system lymphoma. The patients are diagnosed for the ratios IL- lO/IFNy and, optionally, IL10/IL6, during or after their treatment for primary central nervous system lymphoma. A remission is predicted by detecting a decrease in IL-lO/IFNy and, optionally, IL10/IL6 ratios whereas a relapse is predicted by detecting an increase in IL-lO/IFNy and, optionally, IL10/IL6. Accordingly, the invention also relates to a method for monitory the efficacy of a treatment for primary central nervous system lymphoma, in a patient in need thereof, said method comprising the steps of carrying out the diagnosis methods as above defined from a biological sample obtained from a patient having received a treatment for primary central nervous system lymphoma, wherein a decrease in ILlO/IFNy ratio, preferably in both IL-10/IL-6 and IL-lO/IFNy ratios, is indicative of good efficacy of the treatment.

In one specific embodiment of the above method for monitoring the efficacy of a treatment, the treatment is a treatment with methotrexate.

In the following, the invention will be illustrated by means of the following examples as well as the figures.

FIGURE LEGENDS

Figure 1: Correlation of recombinant human IL-10 concentrations measured by CBA and ELISA. Recombinant human IL-10 (rhIL-10) cytokines provided by the CBA (lower graphs) or the ELISA kit (upper graphs) were measured separately by the CBA (left graphs) and the ELISA (right graphs) techniques. This figure represents one out of four experiments.

Figure 2: Correlation of vitreal IL-10 concentrations measured by CBA and ELISA. IL-10 concentration from 30 frozen vitreous samples (13 PIOL, 8 OCL and 9 uveitis patients) were comparatively analyzed at the same time. The horizontal and vertical dashed lines indicate the sensitivity thresholds for ELISA (8 pg/ml) and CBA (2.8 pg/ml), respectively. Figure 3: Thl/Th2 cytokine profile of RD, PIOL, OCL and uveitis samples. The grey circles represent the patients with a retinal detachment (n=l l) considered as the control group, the black triangles refer to PIOL patients (n=17), the grey diamonds symbolize the OCL patients (n=9) and the white squares concern the uveitis patients (n=23). The dashed lines indicate the sensitivity threshold for each cytokine: 2.6, 2.6, 2.8, 2.8, 3, and 7.1 pg/ml for IL-2, IL-4, IL-10, TNFa, IL-6 and IFNy, respectively. All symbols represented under the sensitivity threshold display an equivalent non detectable cytokine concentration.

Figure 4: Vitreous IL-10/IL-6 and IL-lO/IFNy ratios for PIOL, OCL and uveitis samples. Box plots of the distribution of IL-10/IL-6 ratio (A), and ILlO/IFNy ratio (B) for PIOL (n=17), OCL (n=9) and for uveitis (n=23) patient groups.

Figure 5: Combination of the logarithmic conversion of IL-10/IL-6 ratio and IL-lO/IFNy ratio for PIOL, OCL and uveitis patients. The black triangles refer to PIOL patients (n=17), the grey diamonds to OCL patients (n=9) and the white squares concern the uveitis patients (n=23).

Figure 6: Combination of the Logarithmic conversion of IL-10/IL-6 and IL-lO/IFNy ratio in aqueous humor samples from PIOL, OCL and uveitis patients.

(A) The black triangles represent PIOL patients (n=l l), the gray diamonds OCL patients (n=3), and the white squares uveitis patients (n=l l). Kinetics follow-up after intravitreous methotrexate treatment of a remitting patient (B) and of a patient undergoing an initial phase of remission and then a relapse (C). For (B) and (C) size and grey intensity of the marks are correlated to the dates of analyses, the large black circle being the first time- point. Relapse is illustrated by black diamonds.

EXAMPLES

In the following description, all molecular biology experiments for which no detailed protocol is given are performed according to standard protocol.

METHODS Patients and Samples

Sixty patients were enrolled in the study as shown in Table 1.

Table 1. Summary of patient characteristics at diagnosis.

Age Sex

Tissue Final diagnosis Number

Min-max (median) ^§M/"F

: 17 ·^■-: ;^■ 37-89 (75) 5/12

^†OCL 9 45-82 (70) 1 /8

Vitreous

Uveitis 23 ·^· 6-84 (70) 12/1 1

*RD 1 1 54-81 (73) 4/7

^#PIOL, Primary intraocular lymphoma; ^†OCL, Oculocerebral lymphoma;

RD, Retinal detachment; ^§M, male; " F, female.

The patients were distributed in four groups. The first comprised 17 patients with PIOL and the second one 9 patients with OCL diagnosed by cytologic analysis of the vitreous. The third group consisted of 23 patients with diagnosed uveitis and the last one used as a negative control group comprised 11 patients with non-hemorrhagic retinal detachment. Vitreous humour specimens were obtained through a standard three-port pars plana vitrectomy as previously described (Whitcup et al, 1993). Tissue culture medium (balanced salt solution, BSS) enriched with 10% fetal calf serum (FCS) was added to the collection chamber to improve cell viability. An initial 250 μΐ specimen of pure vitreous humour was collected separately into a micro tube for IL-10 quantification only, the remainder was diluted into BSS supplemented with 10% FCS and 2 ml of this preparation were delivered immediately, in a syringe, to the Hematology Laboratory. The vitrectomy cutter was maintained within the vitreous humour to obtain a dilute specimen with minimal disruption. The remainder of the diluted vitreous humour specimen, corresponding to about 20-30 ml, was harvested in a collection cassette and immediately processed for cytological and immunocytochemical analyses. Cytokine Assay

Samples were stored at -80°C and cytokine assays were performed on freshly thawed samples. A minimum of 100 |jL of vitreous sample was used to determine IL-10 concentration using a standard quantifiable sandwich enzyme immunoassay technique (Quantikine®; RD systems, Abingdon, UK) as previously described (Merle-Beral et al., 2004).

For cytokine measurement using the Cytometric Bead Array (CBA) technique, samples were assayed for IL-2, IL-4, IL-6, IL-10, TNFa and IFNy (human Thl/Th2 CBA kit; BD Biosciences), according to the manufacturer's recommendations. Briefly, the six capture bead populations with distinct fluorescence intensities that were coated with cytokine- specific capture antibodies were mixed together in equal volumes. Fifty μL· of each sample and 50μί of PE-conjugated detection antibodies were added to 50μί of mixed-bead populations. The mixture was incubated for 3 hours at room temperature in the dark to form sandwich complexes. The beads were then washed with wash buffer and data were acquired using a LSR II flow cytometer (BD Biosciences). Analyses were performed using Diva and FCAP softwares (BD Biosciences).

Statistical Analysis

PIOL, OCL and uveitis groups were compared by the Mann-Whitney non parametric statistic test or by the Student t-test. P < 0.05 was considered as significant. Results

IL-10 concentration measurement by CBA and correlation with ELISA

A high level of IL10 usually measured by ELISA within pure vitreous or aqueous humor samples is considered as an indirect diagnostic evidence for most of PIOL cases (Cassoux et al., 2007). Before the use of the cytometric bead array (CBA) multiplex technology to analyse the Thl/Th2 cytokine profile of PIOL, OCL and uveitis patients, a comparison between IL-10 results obtained by ELISA and CBA was performed. As shown in figure 1, very high correlations (R =0.994 to 0.999) between IL-10 concentrations measured by the two different techniques were obtained both with the recombinant human IL-10 provided by the CBA or by the ELISA kits. Comparative analyses were next performed for IL-10 concentrations using vitreous samples from PIOL, OCL and uveitis patients (Figure 2).

Interestingly, a good correlation between the two techniques was also obtained (R =0.701). Comparative Thl/Th2 cytokine profiles of PIOL, OCL and uveitis samples

Vitreous samples from 60 patients (Table 1) with PIOL (n=17), OCL (n=9), uveitis (n=23) or traumatic retinal detachment (RD) without haemorrhage (n=l l) were analyzed for IL-2, IL-4, IL-6, IL-10, IFN γ, and TNFcc concentrations by CBA. IL-2, IL-4 and TNFcc were not detected in any sample (Figure 3). RD samples, considered as a control group displayed only a significant level of IL-6 for 82% (9/11) of cases. IL-6, IL-10 and IFNy were detected respectively in 100%, 60%, 48% of uveitis samples. Interestingly, no significant difference was observed between PIOL and OCL cytokine profiles, excepted for IL-10 for which a slight lower concentration was noticed. The mean IL-10 concentration was 724.12 + 676 pg/mL for PIOL and 247.41 + 207 pg/mL for OCL samples (P=0.009). As previously described, the mean IL-10 concentration was higher for PIOL (724.12 + 676 pg/mL) than for uveitis (12.61 + 23 pg/mL) samples (P<0.001). Inversely, the mean IL-6 concentration was lower for PIOL (41.4 + 35 pg/mL) than for uveitis (1184 ± 2394 pg/mL) samples (P=0.018). However, some PIOL and uveitis patients displayed low or high level of IL-10, respectively.

IL-10/IL-6 and IL-10/IFNy ratios for PIOL, OCL and uveitis samples

In order to try to better discriminate PIOL/OCL versus uveitis patients, IL-10/IL-6 and IL- lO/IFNy ratios were calculated (Figure 4). Using the threshold level of detection for negative samples, the mean IL-10/IL-6 and IL-lO/IFNy ratios were both higher for PIOL (IL-10/IL-6, 27.2 + 24 pg/mL; IL-lO/IFNy, 85.98 + 81 pg/mL) and OCL (IL-10/IL-6, 26.68 + 25 pg/mL; IL-lO/IFNy, 33.58 + 30 pg mL), than for uveitis (IL-10/IL-6, 0.15 + 0.22 pg/mL; IL-10/IFNy, 0.54+ 0.66 pg/mL). Interestingly, no significant difference was found between PIOL and OCL mean IL-10/IL-6 or IL-lO/IFNy ratios. It is to notice that the Log calculation of the data allowed us to represent proportional graphic distribution on a linear y-axis instead of a Log axis.

Despite of the significant differences in the mean IL-10/IL-6 and IL-lO/IFNy ratios between PIOL/OCL and uveitis, some of the samples were still overlapping. However, since these overlapping samples were not necessary for the same patient we decided to combine these IL-10/IL-6 and IL-lO/IFNy ratios on a unique graph (Figure 5). Using this analytical strategy, we were able to identify a cluster (upper right) containing exclusively PIOL and OCL samples. In the left parts of the graph, only 1 PIOL sample was colocalized with uveitis samples. We performed the same analysis on aqueous humor from PIOL, OCL, and uveitis patients and obtained similar results (Figure 6A). As aqueous humor can be harvested several times from the same patient, we were thus able to measure cytokine ratios over time during treatment (ie. intravitreous injections of methotrexate). Remission was identified as a decrease in both ratios (see Figure 6B), whereas relapse was correlated with a new increase back in the upper right quadrant of the graph (Figure 6C).

Discussion

The incidence of PCNSL has increased more than ten folds over the past ten years and the incidence of PIOL, as a subtype of PCNSL, has also increased though more slowly, leading to an increasing concern regarding the diagnosis of this affection. Moreover, PIOL is one of the most challenging uveitis masquerade syndromes (Sen et al., 2009). The lack of information concerning this pathology is mainly due to the limited number of patients diagnosed and the low amount of ocular fluids harvested. The present work describes the analysis of the Thl/Th2 cytokine profile in undiluted vitreous samples from PIOL patients using a multiplex based technology that needs only 25 to 50μί of sample. First, we confirmed that a high correlation was obtained between IL-10 concentrations measured by CBA or ELISA, both for a recombinant cytokine or human vitreous. Next, results did not display conventional Thl nor Th2 profile since IL-2, IL-4 and TNFcc were not detected in any sample. In RD control group only a significant level of IL-6 was detected in some of the samples. In contrast, high concentrations of IL-10, and in a lower extent IL-6 and in some cases IFNy were found. Interestingly, cytokine profile of samples from patients presenting an oculo-cerebral lymphoma at diagnosis was similar to PIOL cytokine profile, excepted for IL-10 for which a slight lower concentration was noticed. Despite a differential mean of expression for IL-6 and IL-10 in uveitis vitreous samples, an overlapping was found in some cases. Indeed, 48% (11/23) of the uveitis samples displayed an IL-6 concentration in the same range that PIOL/OCL samples and 38% (10/26) of the PIOL/OCL samples displayed an IL-10 concentration in the same range that uveitis samples. IL-10/IL-6 and IL-10/IFNy ratios displayed significant differences between PIOL/OCL and uveitis samples, but some of them were still overlapping. Moreover, the exact cutoff ratio indicating a positive test result may vary between laboratories and techniques used. Interestingly, the combination of the IL-10/IL-6 and IL- 10/IFNy ratios was highly informative for the discrimination between PIOL/OCL and uveitis samples. Indeed, a PIOL/OCL specific cluster (100%, 25/25) was found, and only 1 over 26 (3,8%) PIOL/OCL sample was colocalized with uveitis samples. Thus, the combination of the IL-10/IL-6 and IL-10/IFNy ratios is very helpful to screen in a first step PIOL-masquerade syndrome among uveitis patients. Moreover, the multiplex technologies offer the opportunity to combine future new diagnostic and prognostic markers since only 25 to 50μί of sample are needed for all these tests performed simultaneously.

Claims

1. An in vitro method of diagnosis of primary central nervous system lymphoma, said method comprising :

a. providing a biological sample from a subject,

b. determining the concentrations of at least IL10 and IFNy biomarkers in said biological sample,

Log([IL10]/[IFNy]) > 0.

2. The method according to Claim 1, further comprising determining the concentrations of IL6 biomarker in said biological sample, wherein said subject is predicted to suffer from primary central nervous system lymphoma when the ratio of concentrations of the biomarkers in said biological samples is such that

Log([IL10]/[IFNy]) > 0 and Log([IL10]/[IL6]) > 0.

3. The method according to Claim 1 or 2, wherein said primary central nervous system lymphoma is primary intraocular lymphoma.

4. The method according to Claim 3, wherein said biological sample is a sample from intra-ocular fluids.

5. The method according to any one of the preceding claims, further comprising the steps of detecting and/or measuring the concentration of one or more of the following biomarkers: sCD25 (soluble CD25), ILIRa, IL-13, IL-16, MIF, MIP-la, Serpin El.

6. The method according to any one of the preceding claims, wherein the biomarker concentrations are determined by cytometric bead array technology.

7. A kit for carrying out the diagnosis method according to any one of claims 1-6, said kit comprising :

a. a detectable binding reagent specific of IL 10 biomarker,

b. a detectable binding reagent specific of IFNy biomarker, c. optionally labelling reagent for detecting said binding reagents and/or appropriate buffers.

8. The kit according to Claim 7, further comprising a detectable binding reagent specific of IL6 biomarker.

9. The kit according to Claim 7 or 8, wherein said binding reagent is an antibody conjugated to a solid support, for example a bead.

10. The kit according to any one of Claims 7-9, wherein said detectable binding reagent is detectable by fluorescent means.

11. The kit according to any one of Claims 7-10, comprising

a. a first set of detectable binding reagents comprising:

i. an antibody directed against IL10 biomarker,

ii. an antibody directed against IFNy biomarker, and,

iii. an antibody directed against IL6 biomarker,

b. a second set of detectable binding reagents comprising:

i. an antibody directed against IL10 biomarker,

ii. an antibody directed against IFNy biomarker, and,

iii. an antibody directed against IL6 biomarker,

wherein each antibody of said second set is conjugated to a fluorochrome.

12. The kit according to any one of Claims 6-11, further comprising detectable binding reagents specific of a biomarker selected from the group consisting of: sCD25 (soluble CD25), ILIRa, IL-13, IL-16, MIF, MIP-la, Serpin El.

13. A method for treating primary central nervous system lymphoma in a patient in need thereof, said method comprising the steps of : a. selecting the patient in need of a treatment for primary central nervous system lymphoma by carrying out the diagnosis method according to any one of Claims 1 to 6, b. administering to said patient a therapeutically effective amount of an appropriate drug for treating said primary central nervous system lymphoma.

A method for monitory the efficacy of a treatment for primary central nervous system lymphoma, in a patient in need thereof, said method comprising the steps of carrying out the diagnosis methods as defined in any one of Claims 1 to 6 from a biological sample obtained from a patient having received a treatment for primary central nervous system lymphoma, wherein a decrease in IL-lO/IFNy ratio is indicative of good efficacy of the treatment.

The method according to Claim 13 or 14, wherein said primary central nervous system lymphoma is a primary intraocular lymphoma.

The method according to Claim 13, 14 or 15, wherein said appropriate drug for treating said primary central nervous system lymphoma is selected from the group consisting of: anti-IL20 antibody, methotrexate, cytarabine, ifosfamide and trofosfamide and their pharmaceutically acceptable salts and esters.