ES2353091B1 - METHOD OF DIFFERENTIAL DIAGNOSIS BETWEEN THE BURKITT LYMPHOMA AND THE DIFFUSE B LARGE CELL LYMPHOMA. - Google Patents

Abstract

Differential diagnostic method between Burkitt lymphoma and diffuse large B-cell lymphoma. # The present invention falls within the field of molecular biology and medicine. Specifically, it refers to a method of obtaining useful data for the differential diagnosis between Burkitt Lymphoma (LB) and Diffuse Large B-cell Lymphoma (LDCBG) by analyzing the truncated forms of the I2PP2A protein, to a method of differential diagnosis between both types of lymphomas and a kit to carry out these methods.

Description

Differential diagnostic method between Burkitt Lymphoma and Diffuse Large B Cell Lymphoma.

The present invention falls within the field of molecular biology and medicine. Specifically, it refers to a method of obtaining useful data for the differential diagnosis between Burkitt Lymphoma (LB) and Diffuse Large B-Cell Lymphoma (LDCBG), to a differential diagnostic method between both types of lymphomas yaunkit to lead to Perform these methods.

Prior art

Burkitt lymphoma (LB) is a very aggressive type of non-Hodgkin lymphoma that usually occurs in children and young people and occasionally in adults. The World Health Organization (WHO) has recognized the existence of three clinical variants: endemic (typical of equatorial Africa and New Guinea), sporadic and associated with immunodeficiencies (Wright. Blood 1999; 93: 758). Its incidence is especially high in Equatorial Africa and Northeast Brazil (Magrath. Am J Pediatr Hematol Oncol 1991; 13: 222-46; Sandlund et al. Leukemia 1997; 11: 743) and accounts for 4050% of lymphomas of non-Hodgkin type in the most industrialized countries (Murphy et al. J Clin Oncol 1989; 7: 18693; Wilson et al. Cancer 1984; 53: 1695-704). LB is one of the fastest growing pediatric tumors (Bouffet et al. Eur J Cancer 1991; 27: 504-9), so that the tumor doubles in size every 24 hours (Kearns et al. Int J Pediatr Otorhinolaryngol 1986 ; 12: 73-84; Ziegler. N Engl J Med 1977; 297: 75-80). Due to these characteristics, this lymphoma follows a very rapid clinical course in the absence of treatment and death occurs within a few months. The LB therefore requires a very fast diagnosis before starting the speci fi c treatment.

LB therapy is based on a very aggressive and intensive chemotherapy treatment that includes different combinations of cyclophosphamide, vincristine, doxorubicin, methotrexate, ifosfamide, etoposide and cytarabine, as well as the intrathecal administration of chemotherapy or the systemic administration of chemotherapeutic agents capable of cross the blood-brain barrier (Divine et al. Ann Oncol 2005; 16: 1928-35; Magrath et al. J Clin Oncol 1996; 14: 92534; Mead et al. Ann Oncol 2002; 13: 1264-74; Pees et al. Ann Hematol 1992; 65: 201-5; Thomas et al. J Clin Oncol 1999; 17: 2461-70) due to the high risk of central nervous system involvement (Bishop et al. Cancer Invest 2000; 18: 574-83) . Unfortunately, the toxicity of this aggressive therapy is enormous due to the side effects that it entails, and includes neurotoxicity, haematological toxicity, severe mucositis, heart disease and infertility (Patte et al. Blood 2007; 109: 2773-80).

Diffuse Large B-cell Lymphoma (LDCBG) is the most common type of non-Hodgkin lymphoma in adults worldwide and accounts for 30-40% of lymphoid neoplasms (Blood 1997; 89: 3909-18). Although it is also a very aggressive mature B-cell lymphoma (Kuppers et al. Nat Rev Cancer 2005; 5: 251-62), the treatment required for its cure is much less aggressive than that used for LB and usually consists of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), together with Rituximab (Bishop et al. Cancer Invest 2000; 18: 574-83). This treatment is insufficient to prevent LB progression while the therapy indicated for LB would imply unnecessary over-treatment of LDCBG, which could cause serious toxic effects (Bishop et al. Cancer Invest 2000; 18: 574-83). Therefore, it is essential to be able to carry out an unequivocal diagnosis of LB and LDCBG.

The diagnosis of LB is based on morphological, cytogenetic-molecular, immunohistochemical and immunophenotyping criteria (Diebold et al. Lyon: IARC Press; 2001. p. 181-4). The presence of a translocation of the C-MYC locus to one of the immunoglobulin loci is an almost indispensable requirement for the diagnosis of LB (Diebold et al. Lyon: IARC Press; 2001. p. 181-4), but no enough because this translocation also occurs in other types of lymphomas (Cogliatti, et al. Br J Haematol 2006; 134: 294-301). In addition, cases of LB have been described in which this translocation is not detected (Braziel et al. Blood 2001; 97: 3713-20; Macpherson et al. J Clin Oncol 1999; 17: 155867; McClure et al. Am J Surg Pathol 2005; 29: 1652-60). Despite all these complementary approaches for diagnosis, it is often difficult to distinguish between LB and other types of B lymphoma. For example, LDCBG may have morphological, immunohistochemical and immunophenotypic traits relatively similar to those of the sporadic variant of LB ( Sporadic LB or LBes) and even present the translocation of the C-MYC locus (Cogliatti, et al. Br J Haematol 2006; 134: 294-301).

Recently, gene expression profiles characteristic of LBes and LDCBG have been determined (Dave et al. N Engl J Med 2006; 354: 2431-42; Hummel et al. N Engl J Med 2006; 354: 2419-30). These studies have shown that the mofological, immunophenotypic and genetic criteria currently used to differentiate a LBes from an LDCBG are frequently inadequate and unreliable. In fact, 17% (Dave et al. N Engl J Med 2006; 354: 243142) or 34% (Hummel et al. N Engl J Med 2006; 354: 2419-30) of the cases identified in these studies with A gene expression profile corresponding to LBes had been previously diagnosed as LDCBG or simply not classified. Although these genomic approaches could theoretically be used for the unequivocal diagnosis of LBes and LDCBG, hybridization of microarrays for the diagnosis of these lymphomas is far from being able to be applied in the clinical routine of most hospitals in the world.

In summary, in the current clinical practice the distinction between LBes and other types of lymphoma depends, in many cases, on only morphological criteria (Troxell et al. Cancer 2005; 105: 310-8), which are not completely reliable and which may lead to a wrong diagnosis. Unfortunately, no easy clinical application methods have been developed that allow a clear delimitation between all cases of LBes and LDCBG (Cogliatti et al. BrJ

Haematol 2006; 134: 294-301) and there is currently no specific molecular marker for any of these lymphomas. This causes pathologists to be faced with the dilemma of how to diagnose a highly proliferative peripheral blast B-cell lymphoma and, worse, when in doubt about which therapeutic approach to use. An accurate diagnosis is necessary, therefore, to prevent the treatment of a “false negative” LB with relatively low doses of chemotherapy, which would not be effective, or the treatment of a “false positive” LB with an intensive chemotherapy regimen, which It would be unnecessary and toxic. Therefore, in daily clinical practice there is a need for some easy method to apply in any hospital that allows unambiguous identification of the LBes and its clear distinction of the LDCBG.

Explanation of the invention.

The present invention refers to a method of obtaining useful data for the differential diagnosis between Burkitt Lymphoma (LB) and Diffuse Large B-Cell Lymphoma (LDCBG), to a differential diagnostic method between both types of lymphomasyaunkit to carry carry out these methods.

In the present invention it is shown how the analysis of the expression of the I2PP2A protein by immunoblot allows to distinguish between tumor samples of LB patients and tumor samples of LDCBG patients. While the complete forms of the I2PP2A protein can be detected in both the LDCBG patient samples and the LB patient samples, the truncated forms of the I2PP2A protein are only detected in the samples of patients with LB. The present invention therefore provides a quick and simple method to perform in the daily clinical practice of any hospital that solves the problem of the difficulty of distinguishing a LB from an LDCBG allowing decision making regarding the therapeutic approach to use.

A first aspect of the present invention refers to a method of obtaining useful data for the differential diagnosis between a LB and an LDCBG comprising:

a) obtain an isolated biological sample from a subject, and

b) analyze the amount of truncated forms of the I2PP2A protein in the sample obtained in (a).

Preferably, the method of obtaining useful data for the differential diagnosis between an LB and an LDCBG further comprises a step:

c) compare the amount detected in step (b) with a reference amount.

Another aspect of the present invention refers to a differential diagnostic method between an LB and an LDCBG comprising the steps (a) - (c) mentioned above which further comprises a step (d) where a quantity of the truncated forms of the I2PP2A protein detected in step (b) greater than the reference amount with which it is compared in step (c) is indicative of the presence of an LB.

The steps (b) and / or (c) of the methods described above can be totally or partially automated, for example, by means of a robotic sensor device for the detection of the quantity in step (b) or the computerized comparison in step (c). In addition to the steps specified above, it can include other additional steps, for example related to the pre-treatment of the sample or the evaluation of the results obtained by these methods.

The World Health Organization has recognized the existence of three clinical variants of the LB: endemic, sporadic and associated with immunodeficiencies (Harris et al. Annals of Oncology 1999; 10: 1419-1432; Wright. Blood 1999; 93: 758) . A preferred aspect of the present invention refers to a method of obtaining useful data for the differential diagnosis between an LB of the sporid variant (LBes) and an LDCBG. Another preferred aspect of the present invention relates to a differential diagnostic method between an LBes and an LDCBG.

The term "diagnosis", as used in the present invention, refers to the ability to discriminate between samples from LB patients and samples from LDCBG, when a method of classifying samples based on the analysis of samples is applied. the amount of the truncated forms of the I2PP2A protein and in the comparison of said detected amount with respect to a reference amount. This detection, as understood by one skilled in the art, is not intended to be correct in 100% of the samples analyzed. However, it requires that a statistically significant amount of the analyzed samples be correctly classified. The amount that is significantly statistical can be established by one skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test or discriminant functions of Fisher Preferably, the confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. Preferably, the value of p is less than 0.1, 0.05, 0.01, 0.005 or 0.0001. Preferably, the present invention allows the disease to be correctly detected in at least 60%, in at least 70%, in at least 80%,

or in at least 90% of the subjects of a certain group or population analyzed.

The I2PP2A protein, also known as "Phosphase protein 2A inhibitor 2" or "nuclear oncogene SET" is encoded in Homo sapiens by the SET gene, with access number in Gene Bank NW_924573.1; SEQ ID NO: 1). Two isoforms of the I2PP2A protein are known that are generated from this gene by alternative processing of its mRNA and that are distinguished by having a different amino-terminal end (Nagata et al. Proc Natl Acad Sci USA 1995; 92: 4279- 4283). Isoform 1, also called TAF-I alpha (NP_001116293.1; SEQ ID NO: 2) is translated from the mRNA with access number in Gene Bank NM_001122821.1; SEQ ID NO: 3). Isoform 2, also called TAF-I beta (NP_003002.2; SEQ ID NO: 4) is translated from the mRNA with access number in Gene Bank NM_003011.3; SEQ ID NO: 5). The molecular size calculated for isoforms 1 and 2 is 33,489 and 32,103 dalton (Da), respectively, although their apparent mobility in polyacrylamide gels with SDS is approximately 40 kDa (Nagata et al. Proc Natl Acad Sci USA 1995; 92: 4279-4283), which suggests the existence of post-translational modifications.

The existence of truncated forms of the I2PP2A protein, which lacks several amino acids of the amino terminus and most of the glutamic and aspartic rich region of the carboxyl terminus, has been described. The relative molecular size of these truncated forms is approximately 20 kDa (Li et al. Biochemistry 1995; 34: 198896; Li et al. J Biol Chem 1996; 271: 11059-62).

The term "I2PP2A protein", as used herein, refers to any of the proteins resulting from the expression of the human SET gene. Therefore, it refers to both isoform 1 or TAF-I alpha, and isoform 2oTAF-I beta, as well as any of its variants, or fragments derived therefrom.

In the sense used in this description, the term "variant" refers to a protein substantially homologous to the I2PP2A protein. In general, a variant includes additions, deletions or substitutions of amino acids. The term "variant" also includes proteins resulting from posttranslational modifications, for example, but not limited to, glycosylation, phosphorylation or methylation.

As used herein, a protein is "substantially homologous" to the I2PP2A protein when its amino acid sequence exhibits a good alignment with the amino acid sequence of any of the isoforms of the I2PP2A protein, that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence of any of the isoforms of the I2PP2A protein, of at least 50%, typically at least 80%, advantageously at least 85%, preferably of at least 90%, more preferably of at least 95%, and, even more preferably of at least 99%. The homologous sequences to the I2PP2A protein can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences.

The term "fragment", as used herein, refers to a portion of the I2PP2A protein or its variants.

The expression "truncated forms of the I2PP2A protein", as used herein refers to those variants or fragments of the I2PP2A protein, which lacks some amino acids from the amino terminus and most of the region rich in glutamic and aspartic of the carboxyl end (Li et al. Biochemistry 1995; 34: 1988-96; Li et al. J Biol Chem 1996; 271: 11059-62). Preferably, the expression "truncated forms of the I2PP2A protein" refers to those variants or fragments of the I2PP2A protein, which have an electrophoretic mobility of less than 30 kDa. More preferably, the expression "truncated forms of the I2PP2A protein" refers to those variants or fragments of the I2PP2A protein, which have an electrophoretic mobility of less than 30 kDa and an isoelectric point (pI) of approximately 5.8. The molecular size and pI of the truncated forms of the I2PP2A protein can be determined by chromatographic and electrophoretic techniques known in the state of the art. Some of the electrophoretic techniques that can be used to determine these characteristics are described elsewhere in this description.

The term "complete forms of the I2PP2A protein", as used herein refers to both isoform 1 or TAF-I alpha, and isoform 2 or TAF-I beta, as well as any of its variants , which have an electrophoretic mobility greater than 30 kDa.

The expression "quantity detection" of a protein, understood as either the I2PP2A protein, either the truncated forms of the I2PP2A protein or the complete forms of the I2PP2A protein, in the sample obtained, as used in the description, refers to the measure of the amount or concentration, preferably semi-quantitatively or quantitatively. The measure can be carried out directly or indirectly. Direct measurement refers to the measure of the amount or concentration of the protein, based on a signal that is obtained directly from the protein, and that is directly correlated with the number of protein molecules present in the sample. Said signal-alaque can also be referred to as an intensity signal - it can be obtained, for example, by measuring an intensity value of a chemical or physical property of the protein. The indirect measurement includes the measurement obtained from a secondary component (for example, a component other than the product of gene expression) or a biological measurement system (for example the measurement of cellular responses, ligands, "tags" or enzymatic reaction products ).

The term "quantity", as used in the description, refers to, but is not limited to, the absolute or relative amount of the protein, as well as any other value or parameter related to or derived from them. . Said values or parameters comprise values of signal intensity obtained from any of the physical or chemical properties of the protein obtained by direct measurement, for example, intensity values of mass spectroscopy or nuclear magnetic resonance. Additionally, said values or parameters include all those obtained by indirect measurement, for example, any of the measurement systems described elsewhere in this document.

The term "comparison", as used in the description, refers to, but is not limited to, the comparison of the amount of truncated forms of the I2PP2A protein of the biological sample to be analyzed, also called the biological problem sample, with an amount of the truncated forms of the I2PP2A protein from a desirable reference sample described elsewhere in the present description. The reference sample can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample. The comparison described in section (c) of the methods of the present invention can be performed manually or assisted by a computer.

The term "reference amount", as used in the description, refers to the amount of the absolute or relative amount of the truncated forms of the I2PP2A protein that makes it possible to discriminate a Burkit lymphoma from other lymphomas. Suitable reference amounts can be determined by the method of the present invention from a reference sample that can be analyzed, for example, simultaneously or consecutively, together with the problem biological sample.

In a preferred embodiment of this aspect of the present invention, the reference amount is obtained from a reference sample from an LDCBG. The reference sample may be, for example, a protein extract obtained from a cell line derived from an LDCBG, or a mixture of protein extracts obtained from several cell lines derived from an LDCBG. Some examples of LDCBG cell lines are for example, but not limited to, Docyl-19, DB, Toledo, Karpas-422 or SUDHL-6. The reference amount can also be obtained, for example, from the normal distribution limits of an amount found in tumor samples obtained from a population of LDCBG patients. This amount can be determined by several well-known techniques, such as the calculation of the average amount of the truncated forms of the I2PP2A protein found in a population of subjects with LDCBG. The amount of truncated forms of the I2PP2A protein is less in LDCBG samples than in LB samples. Depending on the sensitivity of the detection method used, the amount of the truncated forms in LDCBG samples can be considered as undetectable, which can be interpreted as the absence of truncated forms; in contrast, the presence of truncated forms in a problem biological sample is indicative of the presence of a LB.

In another preferred embodiment of this aspect of the present invention, the reference amount is obtained from a reference sample from an LB. The reference sample may be, for example, a protein extract obtained from a cell line derived from an LB, or a mixture of protein extracts obtained from several cell lines derived from an LB. Some examples of LB cell lines are for example, but not limited to, BL2, Akata, Raji, Ramos, DG75 or Mutu-1. The reference amount can also be obtained, for example, from the normal distribution limits of an amount found in tumor samples obtained from a population of LB patients. By means of well-known statistical techniques, the reference amount of the truncated forms of the I2PP2A protein indicative of the presence of a LB can be calculated.

In another preferred embodiment of this aspect of the present invention, the reference amount is obtained from the amount of the complete forms of the I2PP2A protein in the same problem biological sample. An amount of the truncated forms of the I2PP2A protein exceeds, typically, 5%, preferably, 10%, more preferably, 20%, even more preferably, 25%, and even more preferably, 30% of the Detected amount of the complete forms of the I2PP2A protein is indicative of the presence of an LB.

In another preferred embodiment, the reference amount is obtained from the total amount of the I2PP2A protein in the same biological test sample. An amount of the truncated forms of the I2PP2A protein exceeds, typically, 5%, preferably, 10%, more preferably, 20%, even more preferably, 25%, and even more preferably, 30% of the Detected amount of the I2PP2A protein is indicative of the presence of an LB.

In a preferred embodiment, the detection of the amount of truncated forms of the I2PP2A protein is performed by incubation with a specific antibody in an immunoassay. The term "immunoassay", as used herein, refers to any analytical technique that is based on the reaction of conjugation of an antibody with the sample obtained. Examples of immunoassays known in the state of the art are, for example, but not limited to: immunoblot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunohistochemistry or protein chips.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain an antigen binding site that specifically binds (immunoreacts) with the protein. I2PP2A or its truncated forms. Examples of portions of immunologically active immunoglobulin molecules include F (ab) and F (ab ’) 2 fragments that can be generated by treating the antibody with an enzyme such as pepsin. Antibodies can be polyclonal (typically include different antibodies directed against different determinants or epitopes)

or monoclonal (directed against a single determinant in the antigen). The monoclonal antibody may be biochemically altered, by genetic manipulation, or it may be synthetic, possibly lacking the antibody in whole or in part, from portions that are not necessary for the recognition of the I2PP2A protein or its truncated forms and being substituted by others that communicate additional advantageous properties to the antibody. The antibody can also be recombinant, chimeric, humanized, synthetic or a combination of any of the foregoing. A "recombinant antibody or polypeptide" (rAC) is an antibody that has been produced in a host cell that has been transformed or transfected with the nucleic acid encoding the polypeptide, or produces the polypeptide as a result of homologous recombination. Antibodies that recognize the I2PP2A protein or its truncated forms are known in the state of the art, such as those described, but not limited to, in the examples of the present description.

The antibodies used to carry out the methods of the present invention can be polyclonal or monoclonal antibodies capable of recognizing the I2PP2A protein or they can be monoclonal antibodies that recognize only the truncated forms of the I2PP2A protein. Antibodies that recognize the I2PP2A protein can be employed to carry out the methods of the present invention by, for example, but not limited to, immunoblot. Antibodies that recognize only the truncated forms of the I2PP2A protein can be employed to carry out the methods of the present invention, for example, but not limited, by immunoblot, ELISA or immunhistochemistry.

In a preferred embodiment, the immunoassay is an immunoblot or Western blot. To carry out an immunoblot, a protein extract is obtained from an isolated biological sample of a subject and the proteins are separated in a support medium capable of retaining them by electrophoresis. Once the proteins are separated, they are transferred to a different support where they can be detected through the use of specific antibodies that recognize the I2PP2A protein or its truncated forms.

Electrophoresis is an analytical technique of separation of kinetic foundation based on the movement or migration of macromolecules dissolved in a certain medium (electrophoresis buffer solution), through a matrix or cross-linked support as a result of the action of an electric field. The behavior of the molecule is given by its electrophoretic mobility and this by the charge, size and shape thereof. The higher the charge / size ratio, the faster an ion migrates within the electric field. There are numerous variations of this technique depending on the equipment used, support and physical-chemical conditions in which the separation will be carried out. Some variations of this technique are, for example, but not limited to, capillary electrophoresis, paper electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing

or two-dimensional electrophoresis. PAGE electrophoresis can be carried out under denaturing or non-denaturing conditions. Preferably, to detect the amount of the truncated forms of the I2PP2A protein by immunoblot, the proteins obtained from an isolated biological sample of a subject are separated by monodimensional PAGE electrophoresis under denaturing conditions (SDS-PAGE).

Alternatively, proteins obtained from an isolated biological sample of a subject can be separated by two-dimensional electrophoresis or 2D-PAGE. This technique is based on the separation of proteins based on two characteristics: first, the proteins are separated according to their isoelectric point (pI) by isoelectric focusing (IEF) and, secondly, the separation is carried out according to their molecular mass , by electrophoresis under denaturing conditions (SDS-PAGE). The DIGE (Differential In Gel Electrophoresis) technique is based on the protein mapping of the study samples with one of the three fluorochromes (Cy2, Cy3 or Cy5) before separation. The samples are then mixed and separated in a single two-dimensional gel, minimizing experimental variability. Due to the specific marking of each sample, they can be observed individually and made a comparative analysis of the differential expression of proteins, allowing precise quantification.

Once the proteins are separated by electrophoresis, and before detection, the proteins are transferred to a support or to a membrane, for example, but not limited to, PDVF, nitrocellulose or cellulose acetate. This membrane hybridizes with a specific antibody (also called primary antibody) that recognizes the I2PP2A protein

or to its truncated forms. The membrane is then hybridized with an antibody (also called secondary antibody) capable of specifically recognizing the primary antibody and that is conjugated or bound with a marker compound. In an alternative embodiment, it is the antibody that recognizes the I2PP2A protein or its truncated forms that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary. Once the protein is detected, its relative molecular size can be determined by comparing its migration with the migration of a control protein that is detected simultaneously, preferably on the same support, which has a known size.

In another preferred embodiment, the immunoassay is an enzyme-linked immunosorbent assay or ELISA (Enzyme-Linked ImmunoSorbent Assay). The ELISA is based on the premise that an immunoreactive (biological sample antigen or antibody) can be immobilized on a solid support, then bringing that system into contact with a fluid phase containing the complementary reagent that can bind to a marker compound .

There are different types of ELISA. In the direct ELISA or simple two-layer ELISA assay, the solid support is coated with the biological sample and incubated with an antibody that recognizes the truncated forms of the I2PP2A protein, conjugated or bound to a marker compound. In the indirect ELISA, the solid support is coated with the biological sample and incubated with a primary antibody, which recognizes the truncated forms of the I2PP2A protein and then a secondary antibody, which recognizes the primary, conjugated or bound antibody. to a marker compound. In the sandwich ELISA or antigen capture and immunocomplete detection assay, the well is coated with a first antibody that recognizes the truncated forms of the I2PP2A protein, the problem biological sample is applied, so that the truncated forms of the I2PP2A protein they will be retained in the well when recognized by the first antibody, and then a second antibody is applied that recognizes truncated forms of the I2PP2A protein, conjugated or bound to a marker compound.

In another preferred embodiment, the immunoassay is an immunohistochemistry (IHQ). Immunohistochemical techniques allow the identification, on tissue or cytological samples of characteristic antigenic determinants. Immunohistochemical analysis is performed on tissue sections, either frozen or paraffin-derived, from an isolated biological sample of a subject. These membrane sections hybridize with a specific antibody or primary antibody that recognizes the truncated forms of the I2PP2A protein. The membrane is then hybridized with a secondary antibody capable of specifically recognizing the primary antibody and which is conjugated or bound with a marker compound. In an alternative embodiment, it is the antibody that recognizes the truncated forms of the I2PP2 proteins that is conjugated or bound to a marker compound, and the use of a secondary antibody is not necessary.

The term "marker compound", as used herein, refers to a compound capable of giving rise to a chromogenic, fluorogenic, radioactive and / or chemiluminescent signal that allows the detection and quantification of the amount of the I2PP2A protein. or of its truncated forms. The marker compound is selected from the list comprising radioisotopes, enzymes, fluorophores or any molecule capable of being conjugated with another molecule or detected and / or quantified directly. This marker compound can bind to the antibody directly, or through another compound. Some examples of directly binding marker compounds are, but are not limited to, enzymes such as alkaline phosphatase or peroxidase, radioactive isotopes such as 33P or 35S, fluorochromes such as fl uorescein or metal particles, for direct detection by colorimetry, auto-radiography, fl uorimetry , or metallography respectively.

In both LB and LDCBG, the first thing that is detected is a rapidly growing tumor mass that can appear in different regions of the body. For the diagnosis, a biopsy of that mass and blood and bone marrow samples are taken to see if they are also affected.

LB usually presents as a rapidly growing mass that can appear in different regions of the body depending on the lymphoma variant. In most sporadic cases it is an abdominal mass, mainly in the ileocecal region, but it can also be found in the ovaries, kidneys, breast and retroperitoneal region, as well as in lymph nodes in the case of adults (Jaffe et al. Pathology and genetics of tumors of haematopoietic and lymphoid tissues Lyon, France: IARC Press; 2001). In the endemic variant of the LB the mass can also appear in facial bones, digestive tract, ovaries, kidneys and breast. The central nervous system can also be affected in all variants (Bishop et al. Cancer Invest 2000; 18: 574-83). LDCBG patients usually have a rapidly growing mass in either a lymphoid organ or outside them. When it occurs outside of these, it is usually found in the gastrointestinal region, although it can also appear on the skin, bone, testicles, soft tissues, salivary glands, female genital tract, lungs, liver, spleen or central nervous system (Jaffe et al. Pathology and genetics of tumours of haematopoietic and lymphoid tissues Lyon, France: IARC Press; 2001).

Therefore, the term "isolated biological sample", as used in the description refers to, but is not limited to, tissues and / or biological fluids of a subject, obtained by any method known to a person skilled in the art who serve for that purpose. Preferably the isolated biological sample comprises tumor cells, for example, but not limited to, a tumor biopsy. The term "tumor", as used herein, refers to transformed cells that exhibit uncontrolled growth. More preferably, the biological sample is selected from the list comprising: peritoneum, esophagus, stomach, intestine, liver, spleen, pancreas, skin, prostate, testicle, breast, ovary, uterus, cervix, vagina, vulva, kidney, adrenal gland , bladder, urinary tract, bone tissue, muscle, adipose tissue, fibrous tissue, blood vessels salivary glands, liver, spleen, thymus, lung, thyroid, parathyroid, pituitary gland, brain, cerebellum, peripheral nerves, bone marrow, lymph nodes, lymph or blood. The term "subject", as used in the description, refers to animals, preferably mammals, and more preferably, humans.

Another aspect of the present invention is a kit or device comprising the elements necessary to carry out the methods of the present invention.

Preferably, the kit of the present invention comprises the elements necessary for:

a) analyze the amount of truncated forms of the I2PP2A protein in the sample obtained in (a) and

b) compare the quantity detected in step (b) with a reference quantity.

Said kit may contain all those reagents necessary to analyze the amount of the truncated forms of the I2PP2A protein by means of any of the methods described hereinbefore such as, but not limited to, specific antibodies of the I2PP2A protein or its truncated forms, secondary antibodies or positive and / or negative controls. The kit can also include, without any limitation, buffers, protein extraction solutions, agents to prevent contamination, inhibitors of protein degradation, etc. On the other hand, the kit can include all the supports and containers necessary for commissioning and optimization. Preferably, the kit further comprises instructions for carrying out the method of the invention.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention.

Description of the fi gures

Figure 1. Shows an analysis using 2D-DIGE. (A) Experimental design of the strategy used for 2D-DIGE gels and layout scheme with the different fluorophores. (B) Representative 2D-DIGE gel of the gels obtained for the cytosolic fractions of the DOCYL-19 (DLBCL) and DG75 (BL) cell lines.

Figure 2. Shows the detection of a more abundant polypeptide in cytosolic extracts of LBes than in those of LDCBG. (A) A fragment of a 2D-DIGE gel is shown in which the spot or spot 2520 (arrow) is highlighted. In this gel the cytosolic fractions of the cell lines of LBes BL2 and LDCBG Toledo were compared. (B) Abundance of spot 2520 in the samples analyzed relative to the sample of DB cells.

Figure 3. Shows the sequence of the tryptic peptides identified from spot 2520. (A) The specific peptides of isoform 1 and those common to both isoforms are indicated. (B) Sequence of isoforms 1 and 2 in which the amino acids present in the peptides shown in (A) are highlighted in bold. The acidic region of both isoforms is underlined.

Figure 4. Shows the detection of truncated forms of I2PP2A in cytosolic extracts of LB cell lines, but not in those of LDCBG. The detection of the I2PP2A and Tubulin protein (used as a loading control) in cytosolic extracts of the different cell lines indicated in panels (A) and (B) was carried out using the goat anti-I2PP2A sc-5665 antibody (SantaCruz Biotechnology) with the secondary antibody A9452 (Sigma-Aldrich) and the mouse antibody anti-Tubulin T9026 (Sigma-Aldrich) with the secondary antibody A2304 (Sigma-Aldrich). The visualization of the signals was carried out by chemiluminescence.

Figure 5. It shows that the bands detected with the anti-I2PP2A antibody are specific. The detection of the I2PP2A protein in cytosolic extracts of the different cell lines indicated was carried out using the goat anti-I2PP2A sc-5665 antibody (SantaCruz Biotechnology) with the secondary antibody A9452 (Sigma-Aldrich). Previously, 1 μg of the sc-5655 antibody was pre-incubated (+) or not (-) with 5 μg of the competing peptide SC5655P (SantaCruz Biotechnology) before being used for staining the membranes shown. The visualization of the signals was carried out by chemiluminescence.

Figure 6. It shows that I2PP2A is found mostly in the cytosolic fraction of the cell lines of LB (DG75) and LDCBG (Toledo). The proteins present in the indicated subcellular fractions of the DG75 and Toledo cell lines were analyzed by immunoblotting with the anti-I2PP2A sc-5655 antibody and the secondary antibody A9452 (Sigma-Aldrich). The visualization of the signals was carried out by chemiluminescence.

Figure 7. It shows the detection of truncated forms of I2PP2A in the LB DG75 line, but not in that of LDCBG Toledo, by a second anti-I2PP2A antibody that recognizes an epitope other than the previous one. The proteins present in the cytosolic fraction of the DG75 and Toledo cell lines were analyzed by immunoblotting with the anti-I2PP2A sc-27269 antibody and the secondary antibody A9452 (Sigma-Aldrich). The visualization of the signals was carried out by chemiluminescence.

Figure 8. Shows the detection of truncated forms of I2PP2A in cytosolic extracts of LB cell lines, but not in those of LDCBG with a monoclonal antibody. The proteins present in the cytosolic fraction of the indicated cell lines were analyzed by immunoblotting with the anti-I2PP2A mouse monoclonal antibody sc-133138 (SantaCruz Biotechnology) and the secondary antibody A2304 (Sigma-Aldrich). The visualization of the signals was carried out by chemiluminescence.

Figure 9. Shows the detection of truncated forms of I2PP2A in biopsies of LBes patients, but not in those of LDCBG patients. Proteins present in cell extracts from biopsies of LBes (LBes1 to LBes3) or LDCBG (LDCBG1 to LDCBG3) patients frozen in OCT were analyzed by immunoblotting with the anti-I2PP2A sc-5655 antibody and the secondary antibody A9452 (Sigma-Aldrich ). The visualization of the signals was carried out by chemiluminescence.

Examples

The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations to the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application.

Example 1

Analysis of protein expression profile in human cell lines of LB and LDCBG

In order to find a criterion that makes it possible to distinguish between LDCBG and LB in those cases whose diagnosis is confusing with the conventional techniques used in the clinic, we compare the protein expression profiles in different subcellular fractions between LBes and LDCBG cell lines of origin human using a proteomic approach. For this, the cytosolic, nuclear and membrane and organelle fractions of 3 cell lines of LBes (BL2, Ramos and DG75) and LDCBG (Toledo, DB and DOCYL-19) were obtained. Differential protein extraction based on its subcellular location was performed from 12.5x106 cells using the ProteoExtract Subcellular Proteome Extraction kit (Calbiochem). Half of the proteins in each cell fraction were precipitated with methanol / chloroform as previously described (Wessel and Flugge. Anall Biochem 1984; 138: 141143). The proteins were then resuspended in 25 μl of lysis buffer for DIGE [30 mM Tris-HCl (pH 8.5), 7M urea, 2M thiourea, 4% CHAPS and 50 mM DTT] and were incubated at room temperature for 1 h. Protein concentration was determined with the RC DC Protein Assay kit (Bio-Rad). Each of these fractions was compared between the two types of lymphomas in 2D-DIGE two-dimensional gels as previously described (Richard et al. J Proteome Res 2006; 5: 1602-1610) Cytosolic, nuclear and membrane fractions were analyzed independently. Three different two-dimensional (2D) gels were made for the study of each fraction. In each gel, a line of LBes and a line of LDCBG were compared with respect to an internal standard obtained from a mixture of proteins from the 6 cell lines studied. For each analysis, 50 μg of each sample protein was labeled with 400 pmol of Cy3 or Cy5 fluorophores and 50 μg of internal standard protein with 400 pmol of Cy2 fluorophore, according to the instructions described in the Ettan DIGE User (GE Healthcare) manual . The internal standard consisted of 8.33 μg of sample protein corresponding to each of the 6 cell lines included in this example. The experimental design of 2D-DIGE is shown in Figure 1A. The same volume of 2x loading buffer [6 M urea, 2M thiourea, 2% CHAPS, 1% ampholytes pH 3-11NL (GE Healthcare), 10 mM DTT and a minimum amount of bromophenol blue was added ] to the samples and incubated 10 min at room temperature.

The IPG strips (18 cm; pH 3-11NL) (GE Healthcare) were previously rehydrated with 340 μl / ml of rehydration buffer (6M urea, 2M thiourea, 2% CHAPS, 0.5% ampholytes pH 3-11NL , 12 μl / ml DeStreak and a minimum amount of bromophenol blue). In each IPG strip two different samples (labeled with Cy3 and Cy5) and the internal standard (the same amount of proteins in each sample, labeled with Cy2) were loaded, see Figure 1A.

The first dimension (isoelectric focus, IEF) was carried out in an IPGphor (GE Healthcare) applying the following voltage increases: 300 V / h 3 h, gradient 1,000 V 6 h, gradient 8,000V3h, 8,000 V / h 3 ho until 43,000 V totals are reached. After isoelectric focusing, the strips were equilibrated with equilibration solution [50 mM Tris-HCl (pH 8.8), 6M urea, 30% glycerol, 2% SDS and a minimum amount of bromophenol blue]. The strips were equilibrated first with reducing equilibrium solution (with 2% DTT) and then with alkylating equilibrium solution (with 4% iodoacetamide). The second dimension (SDS-PAGE) was performed on 12.5% acrylamide gels (1 mm thick, 26 x 21 cm) in low fluorescence crystals, for 30 min at 2 watts / gel and 4 h at 20 watts / gel, at 20 ° C.

The Cy2-, Cy3-and Cy5-images were obtained on a Typhoon 9400 (GE Healthcare) scanner at the optimal excitation / emission values of each fluorophore and analyzed with the ImageQuant program. Subsequently the gels were stained with silver.

Spot or spot detection, quantification and comparison on each gel was performed using the Differential In-gel Analysis (DIA) and Biological Variation Analysis (BVA) program, modules of version 6.5 of the DeCyder (GE Healthcare) software. Briefly, in DIA, each spot is automatically detected in the images Cy2, Cy3, and Cy5, calculating its normalized abundance. The spot maps are exported to the BVA, matching each among the gels. The selection criteria used to detect spots with differential expression were a variation of the spot volume ratio of 1.5 times and a t-test value of less than 0.05, both in at least 6 of the 9 images obtained.

The main differences were observed when comparing the cytosolic fractions, where several more abundant spots were detected in one type of lymphoma or in the other (Figure 1B). Among all these spots, the number 2520 corresponded to a polypeptide whose abundance in the cytosolic fraction of the BL lines was approximately 2886 times higher than in the same fraction of the LDCBG DB line (Figures 2A and 2B).

Selected differential expression proteins were located on a gel stained with Sypro Ruby (Sigma). The spots of interest were cut by means of a Spot Picker (GE Healthcare) and automatically transferred to a 96-well plate to later be digested in a Brukker digester and identified by MALDI-TOF / TOF mass spectrometry. For the identification of proteins from the peptide spectra obtained, the NCBI database was used using the Mascot 2.1 program.

A total of 10 different tryptic peptides obtained from the digestion of the protein corresponding to spot 2520 were unequivocally identified by MALDI-TOF / TOF mass spectrometry (Figure 3A). Most of these peptides (peptides 4-10) were common to isoforms 1 and 2 of the human I2PP2A protein while the first three peptides, which overlapped with each other, were specific to isoform 1 of this same protein. Figure 3B shows the sequence of isoforms 1 and 2 of I2PP2A, highlighting in bold the amino acids present in the identi fi ed peptides.

It should be noted that the theoretical molecular weight of the two isoforms is approximately 32 and 33 kDa and that their calculated isoelectric points (pI) are approximately 4.1 and 4.2. However, in 2D-DIGE gels, spot 2520 migrates as a protein of apparent molecular weight of approximately 20 kDa and an approximate isoelectric point of 5.8. These differences in the size and pI observed suggested that the form or forms of I2PP2A detected in two-dimensional gels could be truncated forms of the protein in which a large number of negatively charged amino acids should have been lost. Since the carboxyl end of the protein is very rich in glutamic and aspartic residues (underlined in Figure 3), it seems very likely that this region could have been removed from the protein. Supporting this hypothesis, the first published identi fi cation of the I2PP2A protein corresponded to a truncated form of isoform 2 from bovine kidney that lacked the first 16 amino acids and the acidic region present in the carboxyl terminal (Li et al. Biochemistry 1995; 34: 1988-96; Li et al. J Biol Chem 1996; 271: 11059-62).

Example 2

Detection of the I2PP2a protein in human cell lines of LB and LDCBG

The method of the present invention is based on the detection of the I2PP2A protein in protein extracts prepared from patient biopsies. There are two isoforms of this protein: isoform 1, also called TAF-I alpha (Identify Q01105-1) and isoform 2, also called TAF-I beta (Identify Q01105-2). The apparent mobility in polyacrylamide gels with SDS of the complete forms of the I2PP2A protein is around 40 kDa, while that of the truncated forms is approximately 20 kDa (Li et al. Biochemistry 1995; 34: 198896; Li et al. J Biol Chem 1996; 271: 11059-62).

To carry out the detection of the I2PP2A protein, the cytosolic, nuclear, membrane and organelles and 6 cytoskeleton fractions of 6 human LB cell lines (BL2, Akata, Raji, Ramos, DG75 and Mutu-1) were obtained and 5 LDCBG cell lines of human origin (Docyl-19, DB, Toledo, Karpas-422 and SUDHL-6). The fractionation of each cell line was carried out from 12 million cells using the "ProteoExtract Subcellular Proteome Extraction Kit" (Reference 539791 from the Calbiochem company). The proteins present in each fraction were precipitated by adding trichloracetic acid at a final concentration of 10%. The proteins were then resuspended in 50 μl of loading buffer (30 mM Tris-HCl pH 6.5, 7M urea, 2M thiourea, 4% CHAPS and 50 Mm DTT) and incubated at room temperature for 1 h. Protein concentration was determined with the RC DC Protein Assay kit (Bio-Rad).

Using the polyclonal antibody sc-5655 from SantaCruz Biotechnology in an immunoblot assay using the cytosolic fractions of the 6 cell lines of LB and the 2 cell lines of LDCBG, the presence of the complete forms of I2PP2A was detected (around 40 kDa ) on all cell lines. In contrast, the presence of truncated forms of this protein (apparent size less than 30 kDa) was only detected in the LB cell lines (Figure 4A). When this same test was performed comparing the cytosolic extracts of 5 cell lines of LDCBG and 3 cell lines of LB, similar results were obtained (Figure 4B).

The bands recognized by this antibody are specific since pre-incubation of the sc-5655 antibody with an excess of the peptide that it specifically recognizes inhibited the detection of all bands in immunoblot assays (Figure 5).

The expression of I2PP2A in the different fractions obtained using a cell line of LBes (DG75) and another of LDCBG (Toledo) was compared by immunoblotting with the sc-5655 antibody. The results obtained indicated that the majority of this protein is found in the cytosolic fraction, that the great majority of the truncated forms are found only in this fraction and that these truncated forms are not detected in any fraction of the LDCBG line (Figure 6 ).

To confirm the data obtained, an immunoblot was performed with a second polyclonal antibody directed against another epitope of the I2PP2A protein. Incubation with the sc-27269 antibody from SantaCruz Biotechnology also allowed to detect complete and truncated forms of I2PP2A in a cytosolic extract of LB cell lines, while only detecting complete forms in a cytosolic extract of LDCBG cell lines (Figure 7). These data obtained with a different antibody that recognizes a different epitope in I2PP2A confirms, together with the inhibition data of the detection of the bands by the specific peptide, that both the bands that migrate with an approximate size of 40 kDa and the bands that they do it with a size less than 30 kDa correspond to I2PP2A.

Finally, the detection of the I2PP2A protein was carried out using the sc-133138 monoclonal antibody from Santacruz Biotechnology (Figure 8). The proportion of long forms versus that of short forms in Burkitt Lymphoma cells may vary between different preparations of cytosolic extract. However, the presence of short forms is never detected in Diffuse Lymphoma cells, so the presence of short forms is indicative of a Burkitt Lymphoma.

Example 3

Detection of the I2PP2A protein in tumor samples from LBes and LDCBG patients

To apply this method to the clinical diagnosis, frozen tumor OCT samples from 3 LBes patients and 3 LDCBG patients were used. The samples were cut into 15 micron slices with a microtome and 20 slices were used for the test. These slices were stored in a 1.5 ml eppendorf tube and kept in a freezer at -70 ° C until use. To carry out protein extraction and subsequent analysis of I2PP2A expression, tubes containing the samples were placed on ice, 500 microliters of Tissue Extraction Solution (8.5 mM Na2HPO4; 1.5 mM NaH2PO4; 3 mM KCl; 137 mM NaCl; pH 7.4) with a cocktail of protease inhibitors and phosphatases (1 mM Sodium Fluoride, 1 mM Sodium Ortovanadate, 1 mM PMSF, 1 μg / ml Pespstatin A, 1 μg / ml of Leupeptin, 1 μM of Bestatin and 2 μg / ml of Aprotinin) and the sample was pipetted 10 times with a micropipette of 100-1000 μl capacity. Each sample was stirred for 20 seconds with a vortex and centrifuged at 800xg for 3 minutes at 4 ° C. The supernatant was transferred to a new tube and the proteins present in this extract were precipitated with a final concentration of trichloracetic acid of 10%. The proteins were resuspended in 50 μl of loading buffer (30 mM Tris-HCl pH 6.5, 7M urea, 2M thiourea, 4% CHAPS and 50 Mm DTT) and incubated at room temperature for 1 h. Protein concentration was determined with the RC DC Protein Assay kit (Bio-Rad) and these were subsequently analyzed by immunoblotting with the sc-5655 antibody from SantaCruz Biotechnology. The results obtained indicated that both the complete and the truncated forms of I2PP2A were detected in the samples of LBes patients while only the complete forms were detected in the samples of LDCBG patients (Figure 9).

In short, the data presented demonstrate that the detection of truncated forms of I2PP2A would allow distinguishing the LB from the LDCBG by a very easy test to be carried out in any hospital in the world.

Claims (13)

one.

Method of obtaining useful data for the differential diagnosis between an LB and an LDCBG comprising: a) obtaining an isolated biological sample from a subject, and b) analyzing the amount of truncated forms of the I2PP2A protein in the sample obtained in (a ).

2.

Method of obtaining useful data according to claim 1, further comprising a step: c) compare the amount detected in step (b) with a reference amount.

3.

Differential diagnostic method between an LB and an LDCBG comprising steps (a) - (c) according to any of claims 1 and 2, which further comprises a step (d) wherein a quantity of the truncated forms of the I2PP2A protein detected in the step (b) greater than the reference amount with that compared in step (c) is indicative of the presence of an LB.

Four.

Method according to any of claims 1 to 3 wherein the analysis of the amount of the truncated forms of the I2PP2A protein in the sample obtained in (a) is carried out by means of an immunoassay.

5. Method according to claim 4 wherein the immunoassay is an immunoblot.

6.

Method according to any one of claims 1 to 5 wherein the biological sample isolated in step a) is selected from the list comprising: peritoneum, esophagus, stomach, intestine, liver, spleen, pancreas, skin, prostate, testis, breast, ovary , uterus, cervix, vagina, vulva, kidney, adrenal gland, bladder, urinary tract, bone tissue, muscle, adipose tissue, fibrous tissue, blood vessels, salivary glands, liver, spleen, thymus, lung, thyroid, parathyroid gland, pituitary gland , brain, cerebellum, peripheral nerves, bone marrow, lymph nodes, lymph or blood.

7. Method according to any of claims 1 to 6 wherein the LB is of the sporadic variant (LBes).

8.

Kit comprising a specific antibody against the amino terminal region or the central region of the I2PP2A protein for carrying out the methods according to any of claims 1 to 7.

9.

Use of the kit according to claim 8 to analyze the amount of truncated forms of the I2PP2A protein and differentially diagnose Burkitt lymphoma of diffuse large B-cell lymphoma.

SEQUENCE LIST <110> Higher Council for Scientific Research (CSIC) Autonomous University of Madrid <120> Differential diagnostic method between Burkitt Lymphoma and Diffuse Large B Cell Lymphoma. <130> 1641.399 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 6808 <212> DNA <213> Homo sapiens <400> 1  ES 2 353 091 A1    ES 2 353 091 A1    ES 2 353 091 A1    ES 2 353 091 A1    ES 2 353 091 A1   <210> 2 <211> 290 <212> PRT <213> Homo sapiens <400> 2  ES 2 353 091 A1   <210> 3 <211> 2863 <212> DNA <213> Homo sapiens <400> 3  ES 2 353 091 A1    ES 2 353 091 A1    ES 2 353 091 A1   <210> 4 <211> 277 <212> PRT <213> Homo sapiens <400> 4  ES 2 353 091 A1   <210> 5 <211> 2936 <212> DNA <213> Homo sapiens <400> 5  ES 2 353 091 A1    ES 2 353 091 A1   SPANISH OFFICE OF THE PATENTS AND BRAND Application no .: 200900651 SPAIN Date of submission of the application: 09.03.2009 Priority Date: REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: G01N33 / 574 (01.01.2006) RELEVANT DOCUMENTS

Category

Documents cited Claims Affected

X

OUELLET, V, LING, T. H., NORMANDIN, K. et al. Immunohistochemical profiling of benign, low malignant potential and low grade serous epithelial ovarian tumors. BMC Cancer. November 2008, Vol. 8, pages 386-393. ISSN 1471-2407. <DOI: 10.1186 / 1471-2407 / 8/346>. 8

TO

4-6.9

X

CARLSON, S. G., ENG, E., KIM, E.-G. et al. Expression of SET, an inhibitor of protein phosphatase 2A, in renal development and Wilms' tumor. Journal of the American Society of Nephrology. October 1998, Vol. 9, No. 10, pages 1873-1880. ISSN 1046-6673. 8

TO

4-6.9

TO

US 20070105136 A1 (BEA S. M. & OTHERS) 10.05.2007, paragraphs [0114-0124]; examples 18,19. 1,2,6,7,9

TO

SIRMA, S. EKMEKCI, C. G., GROSVELD, G., OZBEK, U. SET (TAF-IB) gene is highly expressed in acute leukemia patients without associated with methylation status. Blood. November 2004, Vol. 104, No. 11, part 2, page 162B, (summary) BIOSIS database. [recovered on 27.11.2011] Recovered from EPOQUE; Accession number PREV200510266959. 4-6,8,9

Category of the documents cited X: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been prepared • for all claims • for claims no:

Date of realization of the report 26.01.2011

Examiner E. Relaño Reyes Page 1/5

REPORT OF THE STATE OF THE TECHNIQUE Application number: 200900651 Minimum documentation sought (classification system followed by classification symbols) G01N Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, TXTE, MEDLINE, BIOSIS, EMBASE, NPL, COMPENDX, INSPEC, XPESP, XPOAC State of the Art Report Page 2/5  WRITTEN OPINION Application number: 200900651 Date of Completion of Written Opinion: 01.21.2011 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims Claims 1-7, 9 8 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims Claims 1-7, 9 8 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/5  WRITTEN OPINION Application number: 200900651 1. Documents considered.- The documents belonging to the state of the art taken into consideration for the realization of this opinion are listed below.

Document

Publication or Identification Number publication date

D01

OUELLET, V. et al. BMC Cancer. November 2008, Vol. 8, pages 386-393. 11.26.2008

D02

CARLSON, S. G. et al. Journal of the American Society of Nephrology. October 1998, Vol. 9, No. 10, pages 1873-1880. 10.1998

D03

US 20070105136 A1 05.10.2007

D04

SIRMA, S. et al. Blood. November 2004, Vol. 104, No. 11, part 2, page 162B. 11/16/2004

D01 discloses an immunohistochemical analysis of different types of ovarian tumors, in order to find markers that distinguish them. Among the genes studied is set, which codes for the I2PP2A protein. In D02, the expression of SET (I2PP2A) is investigated both at the level of mRNA and protein, in developing and adult renal cells, and in different kidney tumor lines. To measure protein expression, polyclonal antibodies were obtained. Thus, it was concluded that the expression of SET is increased in the developing kidney and in the Wilms tumor. D03 optimizes a differential diagnosis method between different types of lymphomas, as well as their prognosis, by means of a "microarray" expression. This analysis includes the study of Burkitt lymphoma (LB) and diffuse large B-cell lymphoma (LDCBG). Being able to distinguish LB from LDCBG is vitally important for choosing the right therapy. Although LB is characterized by the increase in c-myc expression, and the decrease in NF-KB, some LDCBG also have these characteristics, so it is necessary to look for other markers. However, among the genes selected for this purpose, no set is found. In D04, the expression of SET is investigated in patients with acute lymphoblastic leukemia or with acute myeloid leukemia. In both cases an increase in protein expression was found. 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The present application aims at a method of obtaining useful data for the differential diagnosis of sporadic Burkitt lymphoma (LBes) and diffuse large B-cell lymphoma (LDCBG), by analyzing the truncated forms of the I2PP2A protein. In this method, the increase in the amount of truncated forms with respect to the reference value is indicative of the presence of the LB (claims 1 to 3, 6 and 7). The measurement of the truncated forms of I2PP2A can be performed by "immunoblot". (claims 4 and 5). Also included in this application is a kit comprising a specific antibody against the amino terminal region or the central region of I2PP2A (claim 8) and the use of said kit in the differential diagnosis between LB and LDCBG ( claim 9). 1. NEW (Art. 6.1 LP 11/1986)  1.1 CLAIMS FROM 1 TO 7 AND 9 Claims 1 to 7 and 9 meet the novelty requirement.  1.2. CLAIM 8 The object of claim 8 is a kit comprising a specific antibody against the amino terminal region or the central region of I2PP2A. D01 discloses the existence of the commercial antibody sc-5655, specific for the amino terminal region of I2PP2A, and its use in the analysis of the expression of said protein. Therefore, the object of the invention set forth in claim 8, derives directly and without any ambiguity of D01, and claim 8 is not new. State of the Art Report Page 4/5 WRITTEN OPINION Application number: 200900651 2. INVENTIVE ACTIVITY (Art. 8.1 LP 11/1986)  2.1 CLAIMS FROM 1 TO 7 AND 9 Object of the present application, according to claims 1 to 7, is a method of obtaining useful data for the differential diagnosis between Burkitt lymphoma (LB) and diffuse large B-cell lymphoma (LDCBG), which includes the analysis of the amount of truncated forms of I2PP2A, the increase thereof, indicative of the presence of the LB. Likewise, claim 9 relates to the use of a kit comprising a specific antibody against the central or amino terminal regions of I2PP2A, in said method. In none of the cited documents, no relationship has been found between the presence of truncated forms of I2PP2A and Burkitt lymphoma. Accordingly, claims 1 to 7, referring to the indicated method, as well as the use of the kit (claim 9) have inventive activity.  2.2. CLAIM 8 The object of claim 8 is a kit comprising a specific antibody against the terminal or central amino regions of I2PP2A. In D02, the expression of the SET protein (I2PP2A) is investigated. To that end, polyclonal antibodies were produced against the regions or residue 3 to 16, or amino acid 44 to 56 of I2PP2A. It is considered that an expert in the field would try to develop a kit with the antibodies obtained in D02, with a reasonable expectation of success. Consequently, given the present arguments, and the reasons set forth in point 1.2, claim 8 has no inventive activity. State of the Art Report Page 5/5