WO2013190029A1 - Method for the prognosis of a lymphoma and kit for implementing same - Google Patents

Abstract

The invention relates to a method for the diagnosis and the prognosis of the evolution of an especially intraocular lymphoma, comprising measuring the soluble CD25, and to a kit designed for implementing said method.

Description

 METHOD OF PROGNOSIS OF A LYMPHOMA AND KIT FOR ITS IMPLEMENTATION

 ARTWORK

 The invention relates to a new soluble CD25 assay kit, usable in a method of diagnosis and prognosis of progression of lymphoma including intraocular.

The term intraocular lymphoma includes a heterogeneous set of tumor pathologies affecting the eye and manifested in most cases by posterior uveitis resistant to corticosteroid treatment. In all cases, it is a monoclonal lymphoid proliferation with an invasion of a targeted structure of the eye and according to the starting point of lymphoproliferation, there are primitive or secondary intraocular lymphomas.

 Primary Intraocular Lymphoma (IOL) is the primary lymphoma of the central nervous system (PCNSL). Ocular involvement occurs with or without concomitant brain involvement and lymphoma cells preferentially invade the retina or vitreous. In the majority of cases, it is non-Hodgkin's lymphoma, diffuse large B-cell, high grade malignancy.

 PIOL is a rare lymphoma associating ocular and cerebral forms, of difficult diagnosis and putting at risk the patient's vital prognosis. In approximately 80% of cases it is chronic bilateral ocular involvement with subretinal or vitreous infiltration, but unilateral initial presentations are possible. A complication by a cerebral lymphoma is found in about 80% of the cases in the absence of treatment, and when the invasion is bifocal (eye and brain), one retains the term of oculo-cerebral lymphoma (LOC) (Peterson et al , Cancer, 1993 Aug 1, 72 (3): 843-9).

 The clinical symptoms of PIOL are nonspecific, and may mislead the clinician to chronic posterior uveitis. The diagnosis is therefore difficult, often late, and requires a high degree of clinical suspicion on the part of the clinician who will have to be alerted in case of uveitis resistant to treatment with corticosteroids.

Ideally, the treatment of ocular lymphomas should have two goals: to eradicate the intraocular tumor and prevent relapses and brain complications. No optimal therapeutic management has been identified for the moment, the treatments being radiotherapy (irradiation ocular, the tumor being sensitive to treatment, but not curing: relapses are frequent and subsequent brain invasion is possible) or chemotherapy (the main difficulty resides in the lack of penetration of anti-cancer molecules across the blood-brain barrier. ocular), in particular local chemotherapy (intravitreous administration of methotrexate). which allows a long-term remission and can be repeated many times.

As seen above, because of the absence of specific clinical signs, the diagnosis of ocular lymphoma is not easy. Ophthalmological examination will reveal abnormalities in the anterior or posterior segment of the eye. These abnormalities will be explored by anterior chamber puncture (PCA) and the diagnosis will be confirmed by vitrectomy.

 The anterior chamber puncture is minimally invasive and performed first-line when a PIOL is suspected. The yield is low, the volume of the sample being about 10ΟμΙ. It is mainly performed as part of the monitoring of treated PIOLs and for the detection of relapses because the sampling can be done at short time intervals.

 Vitrectomy is a surgical procedure performed under anesthesia, intended to partially or totally remove the vitreous body for diagnostic purposes. In practice, it is performed when the results of the PCA examination evoke a PIOL and must be done at a distance from a corticosteroid therapy in order to avoid a false negative result by lysis of the tumor cells.

 The vitreous is fragmented thanks to a vitreotome placed inside the eye, and the suction is compensated by a fluid intake (hypertonic saline solution, Borate Buffer Saline) to avoid the risk of ocular hypotonia. The first aspiration is considered to be "pure" vitreous, which should be preferred for the diagnostic cytokine assay. The following aspirations report "diluted" vitreous, which can be used for cytological examination.

 Retinal biopsy is performed when vitrectomy is not contributive.

The samples thus taken are then examined, in particular by cytological analysis (performed on vitreous, given the small volume of PCA), This makes it possible to make a definitive diagnosis of PIOL and to start treatment quickly.

 The T or B monoclonal character of the tumor cells can also be demonstrated by molecular analysis (polymerase chain reaction, PCR) making it possible to identify rearrangements in the majority of intraocular lymphoma cases (translocation between chromosomes 14 and 18 or [t (14; 18)], involving IGH and BCL-2 genes, reported in more than 67% of cases).

Interleukin-10 (IL-10) and interleukin-6 (IL-6) assays in aqueous vitreous and aqueous samples are now part of the systematically performed examinations and provide an almost indispensable argument for a diagnosis when intraocular lymphoma is suspected. Indeed, tumor B cells secrete relatively high levels of IL-10 whereas inflammatory reaction cells produce IL-6. The determination of these two cytokines and the determination of their ratio therefore give a good idea of the tumor and inflammatory component involved in the pathological process (Merle-Béral et al., Br. J. Haematol, 2004 Feb; 124 (4): 469-73).

 IL-10 acts as a factor in tumor growth by promoting proliferation of B cell lymphomas (Beatty et al., J. Immunol 1997 May 1, 158 (9): 4045-51), and is produced by lymphoma cells at very high levels.

 Intraocular levels of IL-6 are elevated in uveitis and may be very important in progressive eye infections (toxoplasmic chorioretinitis, acute retinal necrosis related to varicella zoster virus) (Ongkosuwito et al., Invest. Ophthalmol, Vis, Sci 1998 Dec, 39 (13): 2659-65). IL-6 is an important mediator of intraocular inflammation (Hoekzema et al., Curr Eye Eye 1990, 9 Suppl: 207-1 1) because of its active role in the amplification of leukocyte recruitment, and its dosing in the ocular fluids makes it possible to make the differential diagnosis between a panuvéite of infectious, inflammatory or autoimmune origin and a pseudo-uveitis due to a PIOL (Cassoux et al., Invest Ophthalmol, Sci., 2007 Jul ; 48 (7): 3253-9).

In most published studies, the dosage of NL-10 and IL-6 is based on a sandwich-type ELISA test. Several ready-to-use kits are marketed. Assays can be performed on aqueous humor or vitreous. The threshold of IL-10 in the aqueous humor for which the probability of PIOL is probable is 50 ng / mL, with a sensitivity of 89% and a specificity of 93% (assay by ELISA test, Quantikine® kit, R & D System). For vitreous samples, the cutoff is 400 μg / mL with a sensitivity of 80% and a specificity of 99% (Cassoux et al, op.cit.).

 On the other hand, the dilution of the vitreous humor taking place during the vitrectomy is a parameter to be taken into account for the interpretation of the results. The determination of the ratio between IL-10 and IL-6 makes it possible to overcome the effect of the dilution, each cytokine being diluted in the same proportions. An IL-10 / IL-6 ratio of greater than 1 suggests a lymphomatous process (Whitcup et al., Ophthalmol Arch., 1997 Sep; 1 (15): 1,157-60). Thus, according to a study of 35 cases of PIOL and 64 uveitis, the value of the ratio greater than 1 confirmed the diagnosis of PIOL in 74% of cases, with a sensitivity of 74.3% and a specificity of 74.7%.

 The main disadvantage of this technique is the large volume of sample needed for each test.

More recently, the detection of these cytokines has been implemented using a flow cytometry technique, called Cytometric Bead Array ™ (CBA ™ marketed by BD Biosciences). This technique brings a real progress in the diagnosis of PIOL, because it offers the possibility of simultaneously detecting and quantifying several cytokines in a single, low volume sample, unlike the ELISA technique that is usually used as a sample-consuming device.

 It is a "sandwich" type detection, using fluorescent polystyrene microbeads coated with an anti-cytokine antibody (primary antibody), serving as a capture reagent, and a specific detection antibody ( secondary antibody) coupled to a fluorochrome, phycoerythrin (PE), to reveal the complex.

The beads are homogeneous in size and structure but have a specific fluorescence intensity, defined by the manufacturer, easily identifiable with the cytometer by a point cloud well individualized on a dot plot representing the fluorescence in APC (Allophycocyanine) / APCCy7 (Allophycocyanin bound with cyanin-7 (Cy7), emitting by FRET, Fluorescence Resonance Energy Transfer). For the APCCy7, the APC is excited at 633nm. It emits, alone at 665nm, and works as a donor for the Cy7 when linked to it. The Cy7 emits at a wavelength of 767nm. Thus, depending on the amount of fluorescence in the beads and APC / APCCy7 ratio, each point cloud corresponding to a type of ball is associated with an alphanumeric position.

 Each type of ball is coupled to a cytokine-specific primary antibody, so that at each given position corresponds to a cytokine. For example, in the BD-Bioscience Human IL-10 Flex Set kit, the beads coupled to primary antibodies to capture IL-10 are positioned at B7.

 After binding between the ball and the cytokine, the complex is revealed by the secondary antibody coupled to PE, whose fluorescence intensity allows the quantification of the cytokine of interest. This quantification requires the parallel production of a standard range established with a recombinant cytokine.

 Thus, the addition to a sample of several beads of different specificities will allow several cytokines to be assayed simultaneously in a single reaction from a very low sample volume.

Currently, there are no clinico-biological elements to which prognostic value can be attributed. Such a tool could prove useful to the clinician, especially to anticipate the therapeutic management of patients whose prognosis would be bad from the outset.

 The Applicant has thus evaluated the prognostic value of several intraocular soluble factors. The assay of these biomarkers was combined with those of IL-10 and IL-6 using the same flow cytometry (CBA ™) analysis technique, so that all these analytes could be assayed simultaneously. a constant and low sampling volume irrespective of the number of analytes to be assayed, ie a volume of 50 μΙ. Four cytokines were selected: CD25s (or soluble IL-2 receptor), MIP-1a (Macrophage Inflammatory Protein 1 alpha), and RANTES (Regulated upon Activation, Normal T cell Expressed and Secreted, and IFN-gamma Each of these cytokines seems to be of interest for diagnosis and / or prognosis, which is demonstrated for the first time, but the cytokine CD25s is the most interesting.

The interleukin-2 receptor (IL-2) exists in two forms: a membrane form, located on the activated T, B and NK (Natural Killer) lymphocytes, and a free, detectable form in the serum. The membrane form is a heterotrimer consisting of three protein subunits: an IL-2Ra subunit (also called Ag Tac or CD25 or p55), an IL-2R3 subunit (p75) and an IL-2Ry subunit (p64). The α chain is a 55 kDa glycoprotein composed of 251 amino acids, 219 of which are extra cellular, and constitutes the specific recognition site for IL-2. This α chain is the result of an open reading phase leading to the production of a protein of 272 amino acids (SEQ ID No. 1), whose signal peptide of 21 amino acids is cleaved (Kuziel and Greene, J Invest Dermatol 1990 Jun; 94 (6 Suppl): 27S-32S).

 The free form of the receiver consists only of the chain a. This soluble part, called ll-2Ras or CD25s, is a 45kDa protein, detectable in biological fluids and able to bind specifically to IL-2. This protein has three epitopic regions A, B and C.

The Applicant has shown that the amount of CD25s in a patient's eye sample was a factor in anticipating the likelihood of a clinical course of the disease, and could also be used to refine the diagnosis in some cases. Moreover, the Applicant has also shown that certain other factors (IFNy, MIP-1a, RANTES) can also be used for diagnostic purposes and prognosis of PIOLs.

 In view of the constraints related to the amount of sample available in these diseases, the Applicant has therefore developed a kit for detecting CD25s, which is particularly useful by flow cytometry. In particular, the Applicant envisages that this detection kit can be used in parallel with the detection of other analytes, including IL-10 and IL-6. It is indeed important that the detection of many factors can be performed simultaneously, because of the low volume present in the samples taken from patients. The Applicant has, for the first time, and without the results reported in the present application have been expected or predictable, demonstrated that CD25s was an important analyte to be assayed to make a prognosis of evolution of PIOL. The Applicant was therefore the first to ask the question of the interest of developing a test for the detection of this compound, which can be used for small sample volumes, and simultaneously with the detection of other compounds.

Thus, the Applicant has, in particular, developed a detection system that can be used with the CBA ™ system developed by BD Bioscience. The invention thus relates to a detection kit for CD25s comprising a set of beads emitting fluorescence under excitation at an excitation wavelength, said fluorescence emitted by said beads being at a wavelength of emission different from the excitation wavelength, each of the beads comprising, coupled to its surface, a plurality of antibodies directed against the CD25s protein.

 This kit is ideal for detecting CD25s in very small volumes, such as anterior chamber puncture (PCA) or pure vitreous specimen, and can also be used for simultaneous detection of other cytokines. Indeed, if the dosage is performed on the vitreous, it must be pure and undiluted so that the dosages are right but also must be used as little as possible for cytokines because it is necessary to keep equipment for other techniques diagnosis (cytology, immunolabeling, PCR clonality search).

 Thus, if certain documents (US 5,292,636 or WO 87/005912) describe the assay of the CD25s protein in serum, the test used in these documents is an ELISA test, in which a volume (50 μΙ) of sample is used ( see column 24, line 29 of D2). However, since it is possible to take a large volume of serum, it is possible to repeat this ELISA method to measure, independently, the amount of several cytokines. However, the method of US Pat. No. 5,292,636 or WO 87/005912 is not adapted to solving the technical problem of the present invention, which is to be able to assay several cytokines in a very small volume of sample (from vitreous). It is therefore necessary to be able to develop a test to operate in a multiplex.

 The results reported in this application show, for the first time, the presence of CD25s in the vitreous and its role for the diagnosis and prognosis of PIOL. One of the constraints of this disease is the small volume of sampling that can be done, and the fact that it is necessary to be able to dose several metabolites. Without information on the role of CD25s in this disease, it appears that a person skilled in the art would have had no particular reason to manufacture and use such a kit.

WO 2005/031001 describes the use of beads on which antibodies recognizing a CA protein are bound. However, this document provides no indication of the role of CD25s in PIOL, nor of the fact that beads fluorescing under excitation should be used. In a particular embodiment, the wavelength of the fluorescence emitted, after excitation, by each of the balls of the assembly present in the kit is identical.

 In a particular embodiment, the antibodies of the plurality of antibodies directed to the CD25s protein are all identical: this means that each of the beads present in the set of beads contains, on its surface, a plurality of copies of the same antibody directed against CD25s (ie having the same sequence). Thus, in this embodiment, the plurality of antibodies directed against the CD25s protein is a plurality of monoclonal antibodies, i.e., the same monoclonal antibody recognizing CD25s is present, in many instances, at the surface of each of the balls.

 In another embodiment, the antibodies of the plurality of antibodies directed to the CD25s protein are not all identical, but all recognize the same epitope (or domain) of the CD25s protein: this means that each of the beads present in the Bead set contains, on its surface, several identical antibodies against CD25s (that is, having the same sequence).

In a preferred embodiment, the excitation wavelength is less than or equal to 650 nm. Preferably, this excitation wavelength is equal to 633 nm.

 In this embodiment, the emission wavelength (which is detected after excitation) is greater than 655 nm. In particular, it can be equal to 660-665nm or 767-773nm.

 The 633nm wavelength corresponds to a wavelength permitting the excitation of allophycocyanin. This emits at a wavelength around 660nm, detectable with a 665nm filter. When coupled to cyanine-7, cyanine 7 is indeed excited by the fluorescence emitted by allophycocyanin and then emits at 767nm or 773nm, detected at 767nm with a suitable filter. This radiation is based on fluorescence resonance energy transfer (FRET, Fluorescence Resonance Energy Transfer).

In a particular embodiment, said beads emit fluorescence at two different wavelengths. This is the case when the beads contain two fluorophores emitting at these two wavelengths.

 Such beads are described especially in US Pat. No. 7,445,844, US Pat.

6,929,859, US 6,632,526, US 6,599,331, US 6,514,295. In a particular case, said emission wavelengths are 660-665 nm and 767-773 nm, which corresponds to beads containing both allophycocyanin and allophycocyanin coupled to cyanine-7.

 In this embodiment, and as seen above, said beads contain both allophycocyanin and allophycocyanin coupled to cyanine-7, the amount and the ratio being identical for each of the beads.

 Thus, the passage of the beads in a flow cytometer, the excitation at a wavelength of 633 nm, and the collection of the amount of fluorescence emitted by the beads at 655 nm and 767 nm, makes it possible to observe, on a 2D diagram. (abscissa amount of APC fluorescence, ordered amount of fluorescence APCCy7), the beads at a specific position of this diagram.

In a preferred embodiment, said beads are polystyrene microbeads. They preferably have a diameter of between 5 and 10 μηη, more preferably 7.5 μηι.

 In another embodiment, said beads are silica beads (see US 20090068639). In a particular embodiment, said detection kit also contains secondary antibodies capable of binding to CD25s. In the preferred embodiment, these secondary antibodies should be capable of binding to CD25 after binding of this protein to an antibody present on the surface of said beads. It is preferred that these secondary antibodies are bound to a fluorescent compound. The selected fluorochrome preferably has excitation and emission wavelengths different from those of the fluorochromes present in the beads.

 Thus, a fluorochrome having excitation and emission wavelengths of less than 600 nm is preferably chosen. We can choose phycoerythrin (PE), or any other fluorochrome. Those skilled in the art know other usable fluorochromes (listed in particular on wikipedia).

As seen above, it is preferable that the antibodies present on the surface of the beads all recognize the same epitope of the CD25s protein. In particular, it is preferred that they recognize the B epitope of the CD25s protein. Similarly, secondary antibodies all recognize the same epitope or domain of CD25s. In particular, it is preferred that they recognize the epitope (domain) A of the CD25s protein.

Finally, it is preferred that the kit according to the invention also contains a recombinant human CD25s solution, which makes it possible to carry out more precise controls and quantifications.

In a preferred embodiment, the detection kit comprises a) polystyrene microspheres, having in particular a diameter of about 7.5 μηη, containing a mixture of fluorophores Allophycocyanin -

A cyanine-7-linked allophycocyanin, said beads comprising, coupled to their surface, a plurality of murine monoclonal antibodies directed against the human CD25s B epitope

 b) a solution of murine monoclonal antibodies directed against the human CD25s epitope A, linked to phycoerythrin

This kit also optionally includes a recombinant human CD25s solution.

 In a particular embodiment, the beads appear in A4 after passing through a BD FACSCanto ™ II flow cytometer (BD Bioscience), and analysis by appropriate software such as FACSDiva (BD Bioscience). This means that the fluorescence emitted by the beads is similar or identical to the fluorescence emitted by the reference beads 558578 sold by BD Bioscience.

 However, any other beads can be chosen, in particular the balls A5, B4, B5, E5, E8, C8, D8 (BD Bioscience classification). All these beads are available from BD Bioscience. They differ only in the amount and ratio of APC and APCCY7 fluorochromes.

In a preferred embodiment, the detection kit is used in flow cytometry to assay CD25s in a sample obtained from a patient. In a particular embodiment, this sample is a vitreous or other intraocular sample, and at the same time other cytokines are measured.

Thus, the detection kit may also contain at least one other set of beads, said beads comprising, coupled to their surface, a plurality of antibodies recognizing another protein than the CD25s protein, said beads emitting fluorescence under excitation at the same excitation wavelength than that exciting the beads for assaying CD25s, said fluorescence emitted by said beads being

 a) at an emission wavelength different from said emission wavelength of the covered beads of the plurality of antibodies directed against the CD25s protein, and / or

 b) at a different intensity intensity observed for the covered beads of the plurality of antibodies directed against the CD25s protein.

 In particular, said other protein as the CD25s protein is selected from the group consisting of interleukin 6 (IL-6) and interleukin 10 (IL-10).

In particular, beads containing the same fluorochromes (APC / APCCy7) are chosen as the beads intended for the detection of CD25s, but in different proportions and / or quantities.

 Thus, these beads of the second set will be localized, on the 2D diagram (abscissa APC fluorescence quantity, ordered amount of APCCy7 fluorescence), the beads at a specific position of this diagram which is different from that of the beads covered with anti- CD25s.

The kit can thus contain several sets of beads, each intended for detecting and assaying a different protein, the beads of each set having a clean fluorescence profile and different from that of the beads of another set.

 It is preferred when the kit allows simultaneous assay of IL-6 and IL-10 (in addition to CD25s).

 Other sets of beads can be purchased from BD

Bioscience (San Jose, California, USA), for example under the reference 560484 (Human Th1 / Th2 / Th17 CBA Kit which includes beads for the simultaneous detection of IL-2, IL-4, IL-6, IL-2 10, IL-17A, IFN-γ, and TNF), or cytokine by cytokine. For example, mention may be made of reference 558274 (IL-10, beads B7) or 558276 (IL-6, beads A7) from BD Bioscience.

The kit thus described, although it can be used in other applications, has been specifically designed for the implementation of the in vitro measurement (assay) of the amount (concentration) of CD25s in an eye sample in a patient . Indeed, as seen above, the Applicant has specifically developed this kit, in order to perform simultaneous assays of several cytokines in a very small sample, because of the interest of CD25s in the prognosis of primary intraocular lymphoma.

The invention also relates to a prognostic method for intraocular lymphoma in a patient, comprising the step of measuring (in vitro) the amount of CD25s in an eye sample in said patient, a concentration of CD25s greater than 200 pg / ml in said sample being indicative of an unfavorable prognosis of progression of the disease in said patient.

 In a preferred embodiment, this eye sample is a vitreous sample. The concentration measured in vitro is then that actually present in vivo. When the sample is not pure vitreous, it is then necessary to know the dilution to recalculate the effective concentration present in vivo. Thus, if the specimen is vitreous diluted to 50%, the concentration measured in vitro should be multiplied by two to be able to conclude.

 It is understood that the method thus described is carried out in vitro on a sample which has been obtained after sampling from a patient. This method does not include the eye sample collection step.

 This measurement (assay) is especially carried out using the kit described above, which is particularly well suited to such use, having been developed for this purpose.

 Thus this assay is preferably carried out by flow cytometry. Commercially available apparatuses such as two-laser cytometers developed by BD Bioscience can be used.

 Instrument Antibody Detection Secondary Bead Detection Parameters

 BD FACSVerse ™ for PE CBA Red and CBA NIR cytometer

BD Accuri ™ C6 Flow FL2 FL4 (675/25) and FL3 Cytometer ^* (780/60)

 BD FACSArray ™ Yellow Red and NIR

Bioanalyzer

 BD FACSCanto ™ Il for APC PE and APC-Cy ™ 7 Cytometer

BD LSRFortessa ™ for PE APC and APC-Cy7 cytometer BD FACSAria ™ III cell PE APC and APC-Cy7 sorter

 BD FACSCalibur ™ flow FL2 FL4 and FL3

cytometer

 With software such as BD FACSDiva Software, it is possible to isolate the beads corresponding to a particular APC / APCCy7 pair, and to visualize the PE fluorescence of the population identified on a histogram representing the fluorescence intensity on the abscissa and the number of events on the y-axis. The software renders an average fluorescence intensity (MIF) for each tube, corresponding to a concentration determined from a standard range made in parallel.

"Adverse disease prognosis" means a high probability (greater than 50%) that the patient's disease presents at least one of the following changes:

 relapse within one year after stopping treatment

 non-response to treatment (refractory to treatment)

 This means in particular that more than 50% of the patients for whom the concentration of CD25s is greater than 200 μg / ml will have the clinical course mentioned above.

 Similarly, among patients with such a clinical course, more than 50% have a CD25 concentration greater than 200 μg / ml.

 In particular, an amount of CD25s greater than 500 μg / ml in the ocular sample is indicative of a high probability of having a relapse within one year of stopping treatment.

 Similarly, a CD25 concentration of less than 125 μg / ml, or even 150 μg / ml in the ocular sample is indicative of a high probability of complete remission.

 In order to refine this prognosis aspect, it may also be interesting to measure the amount of IL-6 in said sample. Indeed, the presence of high levels of these two inflammatory cytokines is indicative of a poor prognosis for the evolution of the disease.

Thus, an amount of IL-6 greater than 50 μg / ml is indicative of an unfavorable prognosis of disease progression. In particular, an amount of IL-6 greater than 100 μg / ml in the ocular sample is indicative of a high probability of having a relapse within one year of stopping treatment. Finally, it is possible and to consider dosing and measuring the amount of CD25s in a sample of cerebrospinal fluid of a patient, in particular by using the kit thus described, in methods of diagnosis and / or prognosis of a lymphoma of the nervous system. central. This assay is of course performed in vitro. CD25s can indeed be found in samples of this type (Kitai et al, Brain Tumor Pathol, 2013 Jan, 30 (1): 34-9).

 In general, in the methods described above, the in vitro measurement step of the amount of CD25s (CD25s assay) is preferably carried out using a kit as described above.

Description of the Figures

 Figure 1: Representation of the alphanumeric position of the beads carrying the antibodies intended to detect cytokines according to the standard developed by BD Bioscience for the CBA ™ technique, in APC / APC-Cy7 representation

 Figure 2: Summary of clinical information obtained for 25 IOL patients.

 Figure 3: Intravitreal concentration of CD25s measured in patients in complete or refractory treatment-refractory (A.) who relapsed within one year after discontinuation (relapse <1 year) or more than one year after discontinuation of treatment (relapse> 1 year) (B.), figure C. synthesizing figures A. and B. CR: complete remission

Examples

Example 1. Preparation of a Kit for Detection of CD25s by CBA ™

 a) Preparation of the beads coupled to anti CD25 antibodies

 The assay of CD25s in CBA ™ can be performed by coupling beads with specific antibodies.

 For this, were selected non-coupled beads, a pair of monoclonal antibodies each recognizing an epitope different from the CD25s, one purified, intended to be conjugated to the beads, and the other labeled with PE. A standard recombinant of CD25s allows to realize the standard range.

The beads were chosen such that they have a position on the APC / APCCy7 dot plot far away from the other beads that can be used in parallel for the determination of the other cytokines. The choice of balls in position A4 (BD Biosciences, ref 558578) was chosen because this position is far from that of IL-6, IL-10, ΜΙΡΙ -α, RANTES and IFN-v positioned respectively at A7, B7, B9, D4 and El. The cytokine predictive position is shown in Figure 1. The primary antibody chosen is a purified monoclonal mouse antibody

NA The anti-human CD25 (clone M-251) directed against the domain (epitope) B of the protein. The secondary antibody is a human anti-CD25 mouse monoclonal antibody (clone 2A3), directed against the protein domain (epitope) A, coupled to phycoerythrin (excited at a wavelength of 488nm, and emitting a flourescence at a wavelength of 580nm).

 Both antibodies can be purchased from BD Biosciences (San Jose, California) as 555429 and 34101 1 respectively. These antibodies or other antibodies are also available from other suppliers (eg Stemcell, or Miltenyi Biotec).

The primary NA LE anti-CD25 antibody was coupled to the A4 beads according to the manufacturer's recommendations. The principle of the conjugation reaction is to reduce the disulfide bridges on the surface of the polystyrene beads by means of a reducing agent, dithiothreitol. In parallel, the primary amine functions of the antibodies react with a hetero-bifunctional conjugating agent, sulfo-SMCC, and form a highly reactive maleimide derivative. The latter can then form a stable thioether linkage with the thiol functions of the beads via a radical reaction, thus resulting in the covalent anchoring of the antibodies. b) Marking procedure

 The beads coupled to the primary antibody were used for the detection of CD25s, according to the following labeling procedure: for each test, 50μΙ of sample were incubated for 1 hour in the dark with 1 μΙ of conjugated A4 beads beforehand washed and diluted in washing buffer. The anti-CD25 secondary antibody coupled to PE diluted 1/20 was then added. After a 2 hour incubation and a final wash, the sample can be analyzed by cytometer. c) FACSCantoll cytometer analysis - Standard ranges - Linearity

 Each tube containing the reaction medium was analyzed on a cytometer

FACSCantoll ™ 8 colors (BD Bioscience, Becton Dickinson), in manual mode, by acquiring at least 300 events at a "medium" speed. The data was analyzed using BD FACSDiva Software (Becton Dickinson) computer software.

 The analysis strategy first includes conditioning all the beads in a size / structure analysis window. They are then fenestrated in APC and APC-Cy7 so as to identify the maximum A4 beads. This last selection makes it possible to eliminate from the analysis window all the impurities related to the conservation or the doublets of balls generated during the coupling step. The PE fluorescence of the population surrounded by A4 can be visualized on a histogram representing the fluorescence intensity on the abscissa and the number of events on the ordinate. The software renders an average fluorescence intensity (MIF) for each tube, corresponding to a concentration determined from a standard range made in parallel. On the basis of the measurements made on the standard range, it has been observed that there is good linearity for the values between 0 and 10000 μg / ml. Thus, in subsequent assays, the end point of the range should not exceed 10000 pg / ml. As the CD25s assay had never been tested on intraocular samples, it was not possible to predict with certainty what the expected concentration range would be prior to testing patient samples. However, it was considered that the results should be of the same order of magnitude as those of IL-10 and IL-6, for which the maximum value detected in CBA is 5000 μg / ml. Thus, the upper point of the calibration range of CD25s has been set at 8000 pg / ml: this point is in the range of linearity of the curve and makes it possible to scan a very wide concentration range. The results of assaying CD25s greater than 8000 pg / ml were thus made "> 8000 pg / ml".

 A 12-point standard range with more diluted concentrations of

CD25s, ranging from 7.8 μg / ml to 8000 μg / ml, was performed. The results of this range show that the fluorescence intensity corresponding to the very low concentrations of between 0 and 30 μg / ml do not vary proportionally. The detection threshold was thus set at 30 μg / ml. d) Multiplex assay test In order to test the compatibility of the beads thus designed with other commercial beads, to demonstrate the possibility of performing a detection of all cytokines via a single experiment, the reagents for the detection of CD25s, IL-10 , IL-6, and IFN-gamma were combined to see if it was possible to assay all of these cytokines simultaneously, without interfering with their detection.

 For this, a standard range was made from a mixture of recombinant CD25s, IL-10, IL-6, and IFN-γ. Each cytokine is initially at a concentration of 5000 μg / ml in the first tube which constitutes the upper point of the range. It was diluted cascade to obtain the different points of range, each cytokine then being diluted in the same proportions. Finally, the mixture of all the reagents allowing the detection of each cytokine was added to each tube corresponding to a point of range.

It has been observed that the balls do not interfere with each other for their detection since the linearity is good for each calibration line. It can be noted, however, that the signal intensity is lower for the CD25s, even if the linearity remains quite acceptable.

Example 2. Labeling for Multiplex Assay of CD25s, IL-10, IL-6, MIP1-g, RANTES and IFN-γ in CBA ™ in Eye Samples

 This procedure describes the cytokine labeling technique that has been applied. Ready-to-use kits (Flex Set BD ™ CBA kits) were used for the determination of IL-10, IL-6, IFN-γ, MIP-1 g and RANTES . Each kit makes it possible to carry out 50 tests and contains the capture beads coupled to the primary antibody, the secondary antibody, and a standard recombinant in freeze-dried form to be reconstituted at the time of preparation of the standard range. The CBA ™ kits used were developed by BD Biosciences and are sold under the references 558274 (IL-10), 558276 (IL-6), 558324 (RANTES), 558449 (MIP-1 g) and 561515 (IFNv).

For CD25s, the functional A4 beads described above, the secondary anti-CD25 antibody and the standard recombinant in liquid form were used. Buffers required for the dosage recommended by the manufacturer are provided in the CBA ™ Human Soluble Protein Master Buffer Kit, BD Bioscience. a) Preparation of the capture bead solution

 The beads are to be used at a rate of 1 μί per test, ie 50 μί of each ball for a series of 50 tests. They are first washed in 500 μί of Wash Buffer BD wash buffer, centrifuged for 5 min at 1500 rpm. The supernatant is gently aspirated and the beads are resuspended in 2500μΙ of Capture Bead Diluent for Serum / Plasma dilution buffer, ie 50 μΙ per test. The solution is incubated for 15 min at room temperature and in the dark to avoid exciting the fluorochromes present on the beads. b) Extemporaneous Preparation of the PE Secondary Antibody Solution

 For IL-10, IL-6, IFN-γ, MIP-1a and RANTES, 1 μΙ of secondary antibody is required for one test, ie 50 μί of each antibody for a series of 50 tests. Since the minimum necessary amount for a test was not accurately evaluated for CD25s, the same amount of secondary antibody as that chosen for technical development, ie 5 μΙ per test, was used. A total of 250μΙ of anti-CD25 antibodies are required for 50 tests.

 The mixture of all the necessary antibodies for a series of 50 tests represents a volume of 500 μΙ. This volume is diluted quarterly in 2ml of Diluent Detector buffer and 200μΙ of this dilution are necessary for each test. (c) Marking of samples

The marking is carried out on a volume of 50μΙ of sample. A volume of 50 μΙ of the mixture of beads, previously vortexed, is added to the sample and then incubated for 1 hour in the dark and at room temperature. After this incubation, 50 μl of the PE secondary antibody mixture solution is added to the sample. The mixture is incubated for 2 hours in the dark and at room temperature. At the end of this step, the samples are washed to remove excess secondary antibodies. A volume of 1000 μΙ of Wash buffer BD buffer is added, the sample is centrifuged for 5 min at 1500 rpm. The supernatant is gently removed and the bead pellet is resuspended in 300 μΙ of BD Wash Buffer buffer. Samples are ready for cytometer analysis. d) Cytometer analysis

 Before each cytometer run, the sample must be vortexed to resuspend the beads. The analysis must be done at the end of the labeling step to avoid a decoupling between the beads and cytokines that can lead to loss of signal. The samples are analyzed by hand in the cytometer by acquiring at least 300 events at a "medium" speed. The data is analyzed using the BD FACSDiva Software ™ computer software and switched to the FCAP-Array ™ results processing software. e) Technical validation of the test on intraocular samples

 The multiplex cytokine assay of 15 intraocular samples from patients with PIOL (7 PCA and 8 vitreous) and 18 intraocular samples from patients with uveitis was performed on samples stored at -80 ° C for about 2 years. IL-10 and IL-6 were measured initially and simultaneously with the CBA ™ technique. The multiplexed assay of the 6 cytokines was performed after thawing the samples, and the results of IL-10 and IL-6 obtained before and after freezing were compared to see if, on the one hand, the preservation impaired the quality sampling and on the other hand to see if the simultaneous assay of 6 cytokines has an impact on their respective results.

The results are well correlated for IL-10 (R ² = 0.951 1) as well as for IL-6 (R ² = 0.9873) which made it possible to conclude, on the one hand, that freezing did not occur. has not resulted in degradation of the material and secondly, that the simultaneous determination of 6 cytokines does not interfere with the results of each cytokine. The test thus developed is therefore technically valid and provides reliable results.

Example 3. Assay of Different Cytokines in Patients

Apart from IL-10, IL-6 and IFN-gamma, which are regularly routinely assayed in intraocular samples, the dosage of CD25s, RANTES and MIP1-a had never been performed in this study. type of levy. After having developed the combined assay of these 6 cytokines in CBA ™ technique, it was applied to a cohort of patients with intraocular lymphoma (IOL) to see firstly, if these cytokines are detectable in intraocular samples, and on the other hand, to study the clinical significance of the results. a) Patients and samples

 The study included a retrospective cohort of 36 patients with IOL, all from the Pitié-Salpêtrière ophthalmology department, for whom the initial diagnosis was made by cytological examination on a vitreous humor sample. and / or by the assay of IL-10 and IL-6 (assayed in CBA ™ technique, except for samples taken prior to January 2009 for which the assay was performed by ELISA).

 The multiplex assay of the 6 cytokines was performed on the vitreous humor samples corresponding to the diagnostic sample for each of the 36 patients. All samples were stored at -80 ° C and in sufficient quantity to perform a new assay, ie a sampling volume of at least 50μΙ. All these samples were taken between April 2004 and January 2012.

 The cohort included 17 men and 19 women. The average age was 65, with extreme values ranging from 44 to 88 years. For 25 patients, clinical information was obtained from the medical records made available by the Ophthalmology Department.

 In parallel, a "non-PIOL" control cohort was established, comprising 16 vitreous humor samples from patients with a cytokine profile of uveitis (raised intra-ocular IL-6 concentration, low IL-10) and two patients with which IL-10 and IL-6 were not detectable. Clinical information could not be collected for these 18 patients. b) Results of IL-10 and IL-6

 Levels of IL-10 in the vitreous of IOL patients averaged 1787 μg / ml whereas they were 5 μg / ml in the samples of patients in the control group.

The median concentrations of each cytokine were compared between the two groups with a non-parametric Mann-Whitney test, adapted to small series. Median IL-10 concentrations were significantly higher in the IOL group (P <0.05). In contrast, median IL-6 concentrations were higher in the uveitis group (P <0.05). The summary results of the assays are shown in Table 1. IL-10 (μg / ml) IL-6 (μg / ml) Ratio IL-10/1 L-6

IOL Uveitis PIOL Uveitis IOL Uveitis

Median 1 1 10 2 52 207 17.7 0.003

Average 1787 5 265 1406 37 0.008

Difference 1774 4.7 824 2092 44 0.2 type

 Minimum 80 1 6 2 0.9 0

 Maximum 5000 14 5000 5000 21 1 0.5

Table 1: Results of IL-10 and IL-6 in the group of intraocular lymphomas

(n = 36) and uveites (n = 16)

According to a study carried out in the applicant's laboratories, involving 56 glasses in which IL-10 and IL-6 were measured in CBA, the threshold of 37 μg / ml in IL-10 was proposed. to carry the diagnosis of IOL. The minimum IL-10 value detected in this IOL series is 80 μg / ml, which is consistent with this proposed diagnostic threshold. In addition, the calculation of the IL-10 / IL-6 ratio is often considered relevant for providing diagnostic assistance when its value is greater than 1 (Whitcup et al, op.cit.). This ratio has been determined, and it has been observed that it is consistently greater than 1, with the exception of a patient for whom it is 0.9.

 These results thus confirmed the diagnosis of IOL or uveitis initially posed in each group and made it possible to reinforce the technical validity of the test that we have developed, since the initial diagnoses remain unchanged.

The average IFN-gamma concentration in the IOL group is 8 μg / ml whereas it is 3 μg / ml in the uveitis group. Medians are significantly different (P <0.05). The dispersion of results is low in both groups, and only 3 patients in the IOL group have higher concentrations at 21, 37 and 124 pg / ml. However, given the significant overlap of concentration values between the two groups, IFN-γ does not appear to be a relevant parameter for the diagnosis of IOL. c) Results of CD25s, RANTES and MIP-1a Concentrations of RANTES and MIP-1a are higher in IOLs (P <0.05 for RANTES and for MIP-1a) but there is significant overlap of values between the two groups over a range of values between 0 and 50 μg / ml for RANTES and between 0 and 40 μg / ml for MIP-1a. The few patients for whom the values of RANTES and MIP-1a are high correspond to already high IL-10 values. These markers do not provide additional diagnostic assistance.

CD25s concentrations were not significantly different in the two groups of studies, but there was significant heterogeneity of the results in the IOL group. Values range from 30 to 8000 pg / ml in the IOL group, whereas they range from 30 to 837 pg / ml in the uveitis group. High concentrations of CD25s tend to be associated with elevated IL-10 concentrations, but there is no correlation between the two parameters (R ² = 0.4265). It should be noted that the concentrations of CD25s between 840 and 8000 pg / ml are observed only in the context of IOL. Thus, this makes it possible to establish a method for diagnosing intraocular lymphoma in which the amount (concentration) of CD25s in the vitreous or a PCA of a patient is measured, a concentration of greater than 840 μg / ml being strongly indicative of intraocular lymphoma. This method is performed in vitro. This method may also include the treatment step of this patient against this intraocular lymphoma, or a sampling step (PCA or vitreous sampling) prior to the measurement of the concentration of CD25s. This method may also include measuring the amount of IL-10 and / or NL6, optionally simultaneously with the measurement of CD25s. Preferably, this method comprises the simultaneous measurement of the concentrations of CD25s, IL-10 and IL-6 in the sample. One can also evaluate the ratio IL-10/1 L-6 in the context of the implementation of this method. Conclusion

RANTES, MIP-1a and INF-γ do not appear to be more relevant than IL-10 and IL-6 for the diagnosis of IOLs. However, they could be used as second-line to refine a prognosis, but the dosage of IL-10 and IL-6 and calculation of the ratio remain a priori the best elements of the diagnosis. On the other hand, the determination of CD25s at the time of the diagnostic step, especially in combination with ILI O and IL6, makes it possible to refine the diagnosis in case of doubt about the results. obtained with IL-6 and IL-10 alone. Thus, the fact of being able to test a new cytokine (CD25s) in a sample obtained by anterior chamber puncture could also possibly make it possible to reduce the number of vitrectomies, since it makes it possible to remove some doubts whether the results obtained with IL-6 and IL-10 do not allow to conclude with certainty.

Example 4. Interpretation of results: prognostic slope

 For each patient, a limited number of parameters (when they were available) were studied: the diagnosis, the unilateral or bilateral ocular presentation, the presence of concomitant brain damage to the diagnosis, the time to onset of an attack. secondary brain, type of response to first-line treatment (complete response greater than one year, partial response or no response), time to relapse and death after diagnosis. a) Clinical description of 25 patients from the IOL cohort

 Clinical information could only be obtained for 25 patients from the IOL cohort. All of the clinical information obtained is summarized in Figure 2.

Of these 25 patients, 13 have PIOL (no brain damage at diagnosis), 7 have oculo-cerebral lymphoma (LOC), 2 have an ocular location of a relapse of primary brain lymphoma, and 3 Ocular lymphomas secondary to systemic lymphoma (1 follicular lymphoma, 1 ethmoid lymphoma and 1 large cell B lymphoma).

 Of the 12 patients with PIOL, 8 had unilateral, and 5 bilateral, ocular presentations. Three patients developed cerebral invasion within 10 months, 23 months and 24 months after the diagnosis was announced. Five patients developed an ocular relapse of their lymphoma in less than 1 year, whereas only one patient relapsed in more than 1 year. Three patients have been in complete remission for more than one year from the first line of treatment, and 5 patients are refractory to initial treatment (partial response requiring second-line treatment or no response).

Of the 7 LOCs, 2 have unilateral and 4 bilateral ocular involvement. A patient has been in complete remission for more than a year from the first line of treatment, and 2 patients developed an ocular relapse of their cerebral lymphoma treated in a time greater than one year.

 Two patients had an ocular relapse of cerebral lymphoma in remission for 7 years for one and 1 year for the other. The latter died 4 months after the onset of eye relapse.

 Finally, 3 patients have intraocular lymphoma secondary to systemic lymphoma (ethmoidal lymphoma, follicular lymphoma and large cell lymphoma B). For these 3 patients, systemic lymphomas were in remission at the time of ocular relapse. Among them, a patient has concomitant cerebral involvement. In general, it was difficult to know if the patients were still alive at the time of the study. 4 patients had died, but clinical information linking death to lymphoma was not found except for one patient. b) Univariate analysis of the results

 Given the small proportion of clinical information obtained to perform a meaningful multivariate analysis, a non-parametric Mann-Whitney comparison test was performed to determine whether a cytokine increase could be associated with a particular clinical situation. . The concentrations of IL-10, IL-6, CD25s, RANTES, MIP-1a and IFNy were compared in the following groups:

 - PIOL compared to LOC: the 3 patients with secondary ocular lymphoma were excluded to perform this comparison, which was therefore performed on 13 patients with PIOL and 7 patients with LOC.

 - unilateral ocular lesions compared to bilateral lesions, without distinction in the diagnosis of PIOL, LOC or secondary ocular lymphoma, which represents 12 unilateral ocular lesions and 1 1 bilateral lesions.

 - patients in complete remission from the first line of treatment for at least one year (7 patients) compared to patients refractory to treatment, that is to say, partially responding to treatment or requiring multiple lines of treatment (12 patients) .

- Patients with a relapse delay of less than 1 year (5 patients), compared to patients with a relapse delay greater than 1 year (5 patients). c) Comparison between PIOL and LOC The median concentrations of IL-10, RANTES and IFNv are significantly higher in PIOL compared to LOC. This difference is much more marked for IL-10, since the median is 1535 pg / ml in PIOL and 428 pg / ml in LOC. For RANTES and IFNv, even if the concentrations are significantly different, the values remain very low in PIOL and LOC. IL-6 and CD25s show a slight tendency to be increased in PIOL but not significantly. Finally, MIP-1a concentrations are equivalent in both groups (Table 2). d) Comparison between unilateral and bilateral infringements

 There are no significant differences for the different cytokines according to whether the ocular involvement is unilateral or bilateral. IL-10, IL-6 and CD25s appear to tend to be increased in unilateral forms (Table 2).

NS: Not Significant ^* : P <0.05

 Table 2: Comparison of median intravitreal concentrations in the different patient groups studied e) Comparison between patients in complete remission and patients refractory to treatment

There are no statistically significant differences between patients in complete remission for more than one year from the first line of treatment and patients refractory to treatment. However, concentrations of IL-10, IL-6 and CD25s tend to be higher in treatment-refractory patients. RANTES also appears to be increased but to a lesser extent since the concentrations are generally quite low (Table 3). f) Comparison between patients with a relapse delay of less than 1 year and a relapse delay of more than 1 year

 Again, there are no significant differences in cytokine concentrations between patients who relapse within one year of stopping treatment and those who relapse later, with the exception of IFNy, to however, the concentrations are very low. There is, however, a trend of nonsignificant increase in IL-6, CD25s, RANTES and IFNy in patients relapsing early whereas patients in complete remissions for more than one year and patients who relapse late seem to comparable cytokine (Table 3, and Figure 3).

 Table 3: Median Comparison of Intravitreal Concentrations in Different Groups of Patients Studied g) Summary of Results

 Given the small numbers studied each time, it is difficult to observe significant differences between the groups we have formed. However, the order of magnitude of the cytokine concentrations must be taken into account in interpreting these results. Indeed, RANTES, MIP-1a and NFNy are globally always lower than 100 μg / ml whereas IL-10, IL-6 and CD25s can show a significant dispersion of the results with values that can often reach more 1000 μg / ml. Taking into account these elements and with all the reservation related to the limited number of patients studied, it is necessary to interpret the cytokine profiles as a whole, rather than each marker taken one by one.

Note that IL-6, and CD25s (NFNy and RANTES to a lesser extent) vary in the same direction. They tend to be increased at patients with a delayed onset of relapse (1 <year) compared to those whose delay is later. In addition, these same cytokines also tend to be increased in patients responding less well to treatment, compared to those whose complete remission is obtained quickly and sustainably.

 If we combine these results, we also find that the median concentrations of these cytokines in patients with a late relapse delay and in patients in complete remission are close.

 Thus, the increase in inflammatory cytokines at diagnosis seems to give a good indication of the response to treatment. A tumor environment with a significant inflammatory component would rather be associated with a poorer response to treatment.

Thus, the cytokines RANTES, riFNy, MIP-1a and CD25s do not appear to be more relevant than IL-10 and IL-6 for the diagnosis of intraocular lymphomas, but may be useful in complex cases in which second intention, in particular the CD25s, to support a complex diagnosis.

On the other hand, IL-6 and CD25s tend to be increased in patients who relapse within one year after stopping treatment, compared to those with a relapse delay of more than one year. This difference is not statistically significant given the small size studied, but the difference between the medians is significant. RANTES and NFIMy are also increased but to a lesser extent. In addition, patients whose complete remission was achieved quickly and sustainably have similar concentrations of IL-6 and CD25 as those who relapse later.

 In other words, early relapsing patients would experience increased impregnation with pro-inflammatory cytokines, particularly IL-6 and CD25s. It is therefore questionable whether an increased inflammatory response at the time of diagnosis would not play a deleterious role in the host and condition a poorer therapeutic response in the long term.

In addition, IL-10, IL-6, CD25s and RANTES tend to be increased in PIOL compared to LOC. This trend is accentuated by removing the higher points of IL-10, IL-6 and CD25s in the LOC group, which correspond to the same patient whose cytokine profile is rather atypical. This upward trend is also reflected, but to a lesser extent measurement for IL-10 and IL-6 in unilateral ocular forms compared to bilateral lesions. Cytokine concentrations therefore tend to vary according to the unique ocular localization or bilateral or bifocal secondary localization.

 These results give the impression that the cytokine impregnation is greater when the tumor is compartmentalized with the eye, even at the level of only one eye, and that it decreases when the attack extends to another site, as if the immune reaction tended to run out or move to another compartment. To confirm this hypothesis, these cytokines should be assayed in vitreous and CSF at different times of disease progression, at diagnosis and at the time of cerebral invasion to follow the kinetics of these markers as the disease moves to another compartment.

Very interestingly, very high concentrations of CD25s were detected in vitreous patients of PIOL. In general, the origin and role of secretion of CD25s are not fully understood. Some authors attribute a tumor origin while others consider that it reflects the activation of immune cells, including Treg lymphocytes (CD4 + / CD25 + / FOXP3 regulatory T cells) which strongly express the membrane CD25. It is therefore not impossible that the intraocular concentration of CD25s indirectly reflects infiltration by Tregs, which tend to favor tumor progression by inhibiting the immune response. If this hypothesis is true, one can easily understand that high concentrations of CD25s are associated with a poor prognosis for the patient.

Patients who relapse within 1 year of discontinuing treatment tend to have higher IL-6, CD25s, RANTES and IFNy concentrations than patients with late relapse.

Without being bound by this theory, it can be hypothesised that an increase in these cytokines would reflect a significant and deleterious inflammatory response to a good therapeutic response. This inflammatory response would be accompanied by a recruitment of regulatory T cells that would promote tumor progression. Concentrations of IL-10, IL-6 and CD25s also tend to be increased in the purely This could be explained by a more aggressive tumor target and a concentration of immune mechanisms in the eye.

Claims

claims

1. A method of prognosis of intraocular lymphoma in a patient, comprising the step of measuring in vitro the amount of CD25s in an eye sample in said patient, a concentration of CD25s greater than 200 μg / ml in said sample being indicative of an unfavorable prognosis of progression of the disease in said patient.

2. Method according to claim 1, characterized in that the adverse prognosis of progression of the disease in said patient is the probability of having a relapse in the year following discontinuation of treatment (greater than 500 pg / ml).

3. Method according to claim 1, characterized in that the adverse prognosis of evolution of the disease in said patient is the probability of being refractory to treatment.

4. Method according to one of claims 1 to 3, characterized in that one also measures the amount of IL-6 in said sample.

5. A method of diagnosing intraocular lymphoma in a patient, comprising the step of measuring in vitro the amount of CD25s in an eye sample in said patient I, a concentration greater than 840 μg / ml being indicative of a lymphoma intraocular.

6. Method according to claim 5, characterized in that one also measures the amount of IL-10 and / or IL6 in said sample.

7. Method according to claim 6, characterized in that the amount of CD25s, and IL-10 and / or IL-6 are measured simultaneously in said sample.

8. A method of diagnosing central nervous system lymphoma of a patient comprising the step of in vitro measuring the amount of CD25s in a cerebrospinal fluid sample of said patient.

9. Kit for detecting the CD25s protein suitable for implementing a method according to one of claims 1 to 5, comprising a set of beads emitting fluorescence under excitation at an excitation wavelength, said fluorescence emitted by said beads being at an emission wavelength different from the excitation wavelength, each of the beads comprising, coupled to its surface, a plurality of antibodies directed against the CD25s protein.

10. Detection kit according to claim 9, characterized in that said excitation wavelength is less than 650 nm.

1 1. Detection kit according to claim 9 or 10, characterized in that said emission wavelength is greater than 655nm.

12. Detection kit according to one of claims 9 to 11, characterized in that said beads emit fluorescence at two emission wavelengths after excitation at the excitation wavelength.

13. Detection kit according to one of claims 9 to 12, characterized in that said emission wavelengths are detected at 665nm and 767nm.

14. Detection kit according to one of claims 9 to 13, characterized in that said beads are polystyrene microbeads.

15. Detection kit according to one of claims 9 to 14, characterized in that the same monoclonal antibody recognizing CD25s is present in many copies on the surface of each of the beads.

16. Detection kit according to one of claims 9 to 15, characterized in that it also contains secondary antibodies capable of binding to the

CD25s after binding of this protein to said antibodies present on the surface of said beads.

17. Detection kit according to one of claims 9 to 16, characterized in that the antibodies present on the surface of the beads recognize the epitope B of the CD25s protein.

18. Detection kit according to one of claims 9 to 17, characterized in that said secondary antibodies recognize the epitope A of the CD25s protein.

Detection kit according to one of claims 9 to 18, comprising

 a) polystyrene microspheres containing a cyanine-7-linked allophycocyanin-allophycocyanin fluorophore mixture, said beads comprising, coupled at their surface, a plurality of murine monoclonal antibodies directed against the human CD25s B epitope b) a solution of murine monoclonal antibodies directed against the human CD25s epitope A, related to phycoerythrin

 c) optionally a recombinant human CD25s solution

Detection kit according to one of claims 9 to 19, characterized in that it also contains at least one other set of beads, said beads comprising, coupled to their surface, a plurality of antibodies recognizing a protein other than the protein CD25s, said beads emitting fluorescence under excitation at said excitation wavelength, said fluorescence emitted by said beads being

 a) at an emission wavelength different from said emission wavelength of the covered beads of the plurality of antibodies directed against the CD25s protein, and / or

 b) at a different intensity intensity observed for the covered beads of the plurality of antibodies directed against the CD25s protein.

Detection kit according to claim 20, characterized in that said other protein as the CD25s protein is selected from the group consisting of interleukin 6 (IL-6) and interleukin 10 (IL-10).