NZ623642B2 - Lasp-1, a novel urinary marker for transitional cell carcinoma detection - Google Patents

Abstract

Disclosed is the use of LASP-1 in a urine sample obtained from a subject for diagnosing and/or grading transitional cell carcinoma, wherein the presence of LASP-1 above 1 ng/500 µl urine is indicative for transitional cell carcinoma, and an increase of LASP-1 expression correlates with a higher grading of the transitional cell carcinoma. Also disclosed is a method for diagnosing transitional cell carcinoma comprising detecting the presence or absence of LASP-1 in a urine sample obtained from a subject, wherein the presence of LASP-1 above 1 ng/500 µl urine is indicative for transitional cell carcinoma. Also disclosed is a method for grading transitional cell carcinoma comprising determining the level of LASP-1 in a urine sample obtained from a subject, wherein the level of LASP-1 correlates with the grade of the transitional cell carcinoma. ing of the transitional cell carcinoma. Also disclosed is a method for diagnosing transitional cell carcinoma comprising detecting the presence or absence of LASP-1 in a urine sample obtained from a subject, wherein the presence of LASP-1 above 1 ng/500 µl urine is indicative for transitional cell carcinoma. Also disclosed is a method for grading transitional cell carcinoma comprising determining the level of LASP-1 in a urine sample obtained from a subject, wherein the level of LASP-1 correlates with the grade of the transitional cell carcinoma.

Description

WO 83690 PCT/EP2012/074634 LASP-1, a novel Urinary Marker for Transitional Cell Carcinoma ion The invention s to the use of LASP—1 in a urine sample obtained from a subject for 10 diagnosing and/or grading transitional cell carcinoma. The invention furthermore relates to a method for sing transitional cell carcinoma comprising detecting the presence or absence of LASP—1 in a urine sample obtained from a subject, wherein the presence of LASP-1 above 1 ng/500pl urine is indicative for transitional cell oma and a method for grading transitional cell carcinoma sing determining the level of LASP-1 in a urine 15 sample obtained from a subject, wherein the level of LASP—1 correlates with the grade of the transitional cell carcinoma.

In this specification, a number of nts including patent ations and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the 20 patentability of this invention, is herewith incorporated by reference in its entirety. More ically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. 25 The transitional cell carcinoma (TCC) of the urinary bladder is the most common urinary cancer. Men are 3-times more likely to suffer from TCC than women and the dominant age peak is the seventh decade. in 70% gross hematuria is the symptom leading patients to a urological consultation. At the time of diagnosis the majority of TCC are superficial and well treatable by transurethral resection in combination with adjuvant chemo- or immunotherapy 3O [1]. Due to a high recurrence tendency ts need regular follow up cystoscopies, thus making TCC the socio-economic most expensive tumor entity [2].

Diagnosis of TCC currently relies on cystoscopy and urine cytology. Both examinations have limitations: A urethrocystoscopy is invasive, expensive and has a low patients acceptance [3]. 35 Furthermore, cystoscopy has a cy to miss flat lesions, such as carcinoma in situ while urine cytology is prone to miss well differentiated low grade lesions [1, 4]. Both methods are dependent on observer ise. Therefore, much effort has been undertaken to improve the diagnostics of TCC, especially in the follow up care.

So far several diagnostic bladder cancer markers, mostly urine-based, have therefore been developed, e.g. nmp22 or BTA stat [4]. However, in spite of a high sensitivity their specificity is low resulting in unnecessary cystoscopies and biopsies [4]. Therefore, there is a need to identify further biological markers for identifying and grading TCC, in 5 particular novel markers having a high sensitivity and specificity. This need is addressed by the present invention.

Any discussion of the prior art throughout the ication should in no way be considered as an admission that such prior art is widely known or forms part of common 10 general knowledge in the field.

It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to e a useful alternative. 15 Accordingly, in some red embodiments, the present ion relates to the use of LASP-1 in a urine sample obtained from a subject for sing and/or grading transitional cell carcinoma.

Specifically, in a first aspect, the present invention relates to use of LASP-1 in a urine 20 sample obtained from a subject for diagnosing and/or grading transitional cell carcinoma, wherein a LASP-1 level of above 1 μl of urine diagnoses a subject as having transitional cell carcinoma, and an increase of LASP-1 expression ates with a higher grading of the transitional cell carcinoma. 25 In a second aspect, the present invention relates to a method for diagnosing transitional cell carcinoma comprising detecting the presence or absence of LASP-1 in a urine sample obtained from a subject, wherein the presence of LASP-1 above 1 ng/500µl urine is indicative for transitional cell oma. 30 In a third aspect, the present invention relates to a method for grading transitional cell carcinoma comprising determining the level of LASP-1 in a urine sample obtained from a t, wherein the level of LASP-1 correlates with the grade of the transitional cell carcinoma. 35 Unless the t y requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an - 2a - inclusive sense as opposed to an ive or exhaustive sense; that is to say, in the sense of “including, but not limited to” The LIM and SH3 protein 1 (LASP-1) is a member of the LIM protein subfamily which is 5 characterized by a LIM motif and a domain of Src homology region 3. LASP-1 is a focal adhesion protein involved in numerous biological and pathological ses [5-7] and has been linked to an oncogenic function in bladder cancer [6, 7]. Overexpression of the n is observed in several tumor entities including , ovarian and colon cancer [8-10]. In breast cancer a shift of expression s r ce correlates with 10 lymphogenic metastasis and worse overall survival of the patients. Although the function of LASP-1 is not entirely clear, it is known that the protein is involved in cytoskeletal architecture. LASP-1 seems to be constitutively expressed in fast dividing cells like lymphocytes, dendritic cells and monocytes [11] but may also promote ion and proliferation in certain cancer es [5]. Only recently zona occludens 15 protein 2 (ZO-2) was identified as novel LASPbinding partner [18]. It has been found that ZO-2 plays a role in the signal transduction pathway of LASP-1 nucleocytoplasmatic shuttling.

Transitional cell carcinoma (TCC) is also known as urothelial cell carcinoma (UCC) in 20 the art. TCC is a type of cancer that typically occurs in the urinary system: the kidney, urinary bladder, and accessory organs thereof. It is the most common type of bladder cancer and cancer of the ureter, urethra, and urachus. Moreover, it is the second most common type of kidney cancer. In more detail, TCC may form in transitional cells in the lining of the renal pelvis (the part of the kidney that collects, holds, and drains urine) and 25 in this case may be classified as kidney cancer. However, TCC has to be held distinct from renal cell carcinoma (RCC) (or hypernephroma). RCC is a kidney cancer that originates in the lining of the proximal uted tubule, the very small tubes in the kidney that filter the blood and remove waste products. RCC is the most common type of kidney cancer. RCC thus is often referred to in the art as kidney cancer. In 30 accordance with the invention, TCC is preferably a tumor of the urinary bladder.

The term “urine” as used herein designates a typically e liquid by-product of the body that WO 2013/083690 PCT/EP2012/074634 3 is secreted by the kidneys through a process called urination and is excreted through the urethra. Typically, about 10 mL urine are required for the uses and methods of the invention, although some lower as well as higher values are feasible. In any case, it is recommended that amounts higher than 1 ml are used. If the urine proceeds directly to the uses and methods of the ion it is preferred that the urine is not stored longer than 24h at room temperature. In case it should not be possible or desirable that the urine proceeds to the uses and methods of the invention within 24h it is able to centrifuge the urine, discard the supernatant and store the frozen urine cell pellet until the (resuspended) cell pellet proceeds to the uses and methods of the invention (cf. the examples herein below for further details). 10 The term “diagnosing” as used herein is directed to the identification of a subject having a disease. In ance with the invention the disease is transitional cell carcinoma.

The term “grading” as used herein is directed to the identification of the degree of cell 15 sia of a tumor cell in a subject which has been diagnosed as to have a tumor. The most commonly system used for g tumors is the system according to the guidelines of the American Joint Commission on Cancer. As per these guidelines, the following grading ries are distinguished: GX (grade cannot be assessed), G1 (well-differentiated; low grade), GZ (moderately differentiated; intermediate , G3 (poorly differentiated, high 20 grade); G4 (undifferentiated, high grade). In accordance with the ion the tumor is a transitional cell carcinoma.

The term “subject” in accordance with the invention refers to a mammal, preferably a domestic animal or a pet animal such as horse, , pig, sheep, goat, dog or cat, and most 25 preferably a human.

Generally LASP-1 levels of above 1 ng/SOOuI of urine pellet allow the conclusion that a subject has a transitional cell carcinoma. In accordance with the first embodiment of the invention it is thus preferred that a LASP—1 level of above 1 ng/500ul of urine pellet diagnoses 3O a subject having a tional cell carcinoma. It is also preferred with regard to the first embodiment of the invention that an increase of LASP-1 expression correlates with a higher grading of the tional cell carcinoma.

The LASP-1 levels described herein may be determined, for example, by using a “molecule 35 binding to LASP-1” and preferably a “molecule specifically binding to LASP—1”. A molecule binding to LASP—1 ates a molecule which under known conditions occurs WO 2013/083690 PCT/EP2012/074634 4 predominantly bound to LASP-l. Preferably, the dissociation constant KB of the complex formed by said le and the target molecule is less than 10'3 M. More preferred KD is less than 10’5 M, yet more preferred less than 10'7 M, and most preferred less than 10'9 M. Binding involves in general interaction(s) between one or more moieties or functional groups of the binding molecule and one or more moieties or functional groups of LASP—1, wherein said interaction may comprise one or more of -charge interactions; charge—dipole interactions; dipole—dipole interactions, wherein said dipoles may be permanent, induced or fluctuating; hydrogen bonds; and hydrophobic interactions. Hydrogen bonds and interactions involving a permanent dipole are of particular relevance in the sense that they confer 10 specificity of binding by their directional character.

Binding may be unspecific, for example by interaction with a group such as a charge or a dipole, which may be present many times at the e of the target molecule. Preferably, g is specific, i.e., it occurs at a defined site of LASP-1 and goes along with the ion 15 of a network of several ct and specific interactions. Specific binding may occur with hardly any change of the conformation of the les involved (“key—in-lock”), or it may involve conformational s of one or both of the binding partners (“hand-in—glove” gm). One or more g molecules may bind to LASP—l. if more than one test molecule binds LASP-l, the binding molecules may either bind at the same site or at 20 overlapping sites, giving rise to itive binding, or bind to distinct sites such that no interference between the molecules binding to distinct sites of LASP-1 occurs. Accordingly, the term “complex” embraces binary complexes of the type (LASP-i):(binding molecule) and ternary complexes of the type (LASP—1):(first binding molecule):(second binding molecule) as well as complexes of a LASP-1 with more than two binding les. In cases, where more 25 than one g molecule is e of binding to LASP-l, both binary and ternary (and higher order) complexes may be formed.

A “molecule binding to LASP-1” is preferably an antibody, aptamer, fragment or derivative thereof specifically recognizing LASP—‘l, or fragment or epitope thereof. Said dy may be 30 a monoclonal or a polyclonal antibody. The antibody is preferably a monoclonal antibody.

The term “antibody” as used herein includes monoclonal antibodies, polyclonal antibodies, single chain antibodies, or fragments thereof that specifically bind LASP-1, or fragment or epitope thereof, also including bispecific antibodies, tic antibodies, antibody fragments, 35 such as Fab, Fd, F(ab)2, Fv or scFv fragments etc., or a ally modified derivative of any of these. Monoclonal antibodies can be prepared, for example, by the techniques as originally WO 2013/083690 PCT/EP2012/074634 5 described in Kohler and Milstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals with modifications developed by the art. Furthermore, dies or fragments thereof to the aforementioned peptides can be ed by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodies are obtained by the phage y technique, surface n resonance as employed in the BlAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the peptide or polypeptide of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97—105; 10 Malmborg, J. lmmunol. Methods 183 (1995), 7—13). The production of chimeric antibodies is described, for example, in WO89/09622. A r source of dies to be utilized in accordance with the present invention are so-called xenogenic antibodies. The general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735. 15 Antibodies to be employed in accordance with the invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), ion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the 20 amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

The term “monoclonal” or “polyclonal antibody” (see Harlow and Lane, (1988), loc. cit.) also 25 s to derivatives of said antibodies which retain or essentially retain their binding specificity. s particularly red embodiments of said derivatives are ied further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human nt region. 30 The term "scFv fragment" (single-chain Fv fragment) is well understood in the art and preferred due to its small size and the possibility to recombinantly produce such fragments.

Preferably, the dy, aptamer, fragment or derivative thereof according to the invention specifically binds the , or fragment or epitope thereof whose presence or absence is 35 to be monitored.

WO 2013/083690 PCT/EP2012/074634 6 The term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (poly)peptides or epitopes of similar structures. Cross—reactivity of a panel of antibodies etc. under investigation may be tested, for example, by assessing g of said panel of antibodies etc. under conventional conditions (see, e.g., Harlow and Lane, (1988), loc. cit.) to the peptide of interest as well as to a number of more or less (structurally and/or onally) closely related (poly)peptides. Only those antibodies that bind to the peptide/protein of st (i.e. LASP-1, or nt or epitope thereof) but do not or do not essentially bind to any of the other (poly)peptides which are preferably expressed by the 10 same cells as the (poly)peptide of interest, are considered specific for the (poly)peptide/protein of interest and selected for further studies in accordance with the use and method of the invention. it is particularly preferred that said antibody or antibody g n is or is derived from a human antibody or a humanized dy. 15 The term "humanized antibody" means, in accordance with the present invention, an antibody of man origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and ably all 6 CDRs have been replaced by CDRs of an dy of human origin having a desired icity. Optionally, the non—human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human 20 antibody. Methods for the production of humanized antibodies are described in, e.g., EP—A1 0 239 400 and W090/07861.

Aptamers are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. Aptamers have been selected which bind nucleic acid, 25 proteins, small c compounds, and even entire organisms. A database of aptamers is maintained at http://aptamer.icmb.utexas.edu/.

As is evident from examples below, the inventors have unexpectedly identified LASP—1 as a marker for transitional cell carcinoma in urine samples. To the best knowledge of the 30 inventors, the use of LASP-1 as a marker for TCC overcomes several limitations of the diagnostic of TCC available from the prior art.

Cystoscopy and urinary cytology have an observer dependency and limitations. Furthermore, cystoscopy has a low patients acceptance. Current diagnostic markers cannot replace 35 cystoscopy and/or urinary cytology, as they have a low specificity resulting in a high number of false positive results and unnecessary examinations, such as nmp22 or BTA stat [4].

WO 2013/083690 PCT/EP2012/074634 LASP-1 expression is detected in fast proliferating cells, such as the mucosa of the stomach

[11], in the tested cancer entities medulloblastoma, metastatic breast, ovarian and colon cancer [8—10, 14] and - as demonstrated now by the inventors herein by immunohistology - in urotheiium of bladder and ureter. As it is evident from the examples below, in TCC an increased sion of LASP-1 is noted throughout the urotheiium but this overexpression was only te when investigated by immunohistochemistry.

Measurement of LASP-1 content in urinary cell pellets is highly sensitive for TCC. A sensitivity 10 of 83.1 % and a specificity of 85.3 % enable this protein to be a used for the detection and grading of TCC. According to the knowledge of the inventors such good performance was not found for any prior art marker for TCC. The high specificity advantageously reduces false positive results and, hence, unnecessary cystoscopies and es and turns this marker into a striking new tool suitable to complement or ment the current gold rd of 15 cystoscopy and urine cytology.

Aithough the inventors do not wish to be bound by this hypothesis, it is believed that the reason for the increased LASP-1 content in the urine of patients with TCC is an increased shedding of tumor cells into the urine cells. An enhanced loss of e-cadherin and other 20 adhesion molecules is well known in TCC [15] and in agreement with a tower detection rate for well differentiated tumors compared to more aggressive high grade tumors in urine cystoiogy [1]. Therefore, it is tentative to speculate that LASP-1 further raises tumor cell shedding into the urine by a disassembly of focal adhesion contacts. indeed, the examples herein show an impaired cell migration and increased adhesion after LASP-1 knockdown in 25 bladder cancer cells. Therefore, increased LASP-1 content of y cell pellets in TCC may be explained by a higher t of tumor cells.

It should not go unnoted, that the total protein content of the cells in the urine sample (Le. a urine cell pellet in accordance with the examples described herein) was not necessarily 3O predictive for TCC. e low protein content in the urine sample few tumor patients showed high LASP-1 levels while some control volunteers with high overall protein content showed no LASP-1 staining in the Western Blots. Aithough, overall, tumor patients exhibited a higher number of cells in urine samples nced by bigger urinary cell pellets) this difference was not e enough for ion of TCC. 35 Initially, the finding of an nt expression of LASP-1, both in healthy urotheiium and WO 2013/083690 PCT/EP2012/074634 8 TCC, was surprising. r, a strong and therefore easily visible overexpression of LASP- 1 may not be necessary for its oncogenic function. For example, in breast cancer the pro— metastatic effect is not ed by overexpression but by a nuclear localization of LASP-1 [8], and [6] reported a discrepancy between LASP mRNA expression and actual protein level.

The s of [6] hypothesize that LASP protein levels might be decreased by increased ubiquitination and proteolysis in some tumor cell lines. This g might explain that in some murine bladder cancer cell lines LASP-1 expression was decreased while other studies report an increase of LASP—1 expression [16] [17]. The constitutively high sion of LASP—1 in highly proliferating cells may mask an increase or shift in localization pattern [11]. 10 In summary, the inventors have unexpectedly found that measurement of urinary LASP—1 is ‘ an ideal marker for the detection and grading of TCC. The use of LASP—1 as marker for TCC can replace or augment the current gold standard for TCC—diagnosis, namely cystoscopy and urine cytology. The detection of LASP-1 in urine samples, may further help to reduce the 15 s of cystoscopical evaluations in follow up ations, e.g. for the grading of TCC.

The present invention furthermore relates to a method for diagnosing transitional cell carcinoma comprising detecting the presence or absence of LASP-1 in a urine sample obtained from a t, wherein the presence of LASP-1 above 1 ng/500ul urine pellet is 20 indicative for transitional cell carcinoma.

Traces of LASP-1 may also be found in urine samples obtained from healthy subject (i.e. in ular a subject not having transitional cell carcinoma). Though, the presence of LASP-1 above 1 ng/500ul of urine pellet is to the best knowledge of the inventors indicative for 25 transitional cell carcinoma.

The present invention also relates to a method for grading transitional cell oma comprising determining the level of LASP-1 in a urine sample obtained from a subject, wherein the level of LASP-1 correlates with the grade of the transitional cell carcinoma. 30 By correlation analysis of LASP-1 content with the grading of transitional cell carcinomas the inventors revealed a clear increase of LASP-1 sion with higher grading. Thus, the LASP—1 ts ate with the grading of the transitional cell carcinoma. The high urinary LASP—1 levels in increased tumor grades indicate that aggressive tumors t a stronger 35 LASP-1 expression. Migration and adhesion experiments underline this fact.

WO 2013/083690 PCT/EP2012/074634 9 In accordance with a preferred embodiment of the invention the urine sample is unprocessed or processed (e.g stabilized) urine, a urinary cell pellet or a resuspended urinary cell pellet.

The urine sample may be stabilized, for example, by adding substances which prevent ar and in particular protein degradation within the urine sample. Such substances are well-know in the filed of medical stics.

As it is evident from the examples herein below, a urinary cell pellet may, for e, be obtained by transferring 10mL urine to a 15mL tube and centrifuging at 3.3009 for 10 minutes to pellet the cells. The supernatant is poured off carefully so as not to disturb or dislodge the 10 cell pellet. A resuspended urinary cell pellet (independent of quantity of the pellet) may, for example, be obtained by resuspending each pellet in 200 pl Laemmli sample , heating at 95°C for 5min. The suspension may be stored at -20°C until further processing.

In accordance with a further preferred embodiment of the invention the urine sample has 15 been obtained from a subject not having a urinary tract infection.

The use of urinary LASP-1 measurement for detection or grading of TCC may give a false positive result if the urine sample is contaminated with leukocytes (i.e. a specific cell—type defending the body against infectious disease and foreign material). Leukocytes have a high 20 cellular LASP—1 t. Therefore urine samples obtained from subjects with urinary tract infections are preferably excluded. The ts’ urine may then preferably be obtained when the ion was successfully treated. in ance with another preferred embodiment of the invention the urine sample 25 comprises less than 250 erythrocytes per ul unprocessed urine, preferably less than 200 ocytes per ul essed urine and more preferably less than 150 erythrocytes per ul unprocessed urine.

Also contamination with erythrocytes (i.e. red blood cells) above 250 ocytes per ul of 30 unprocessed urine may give false positive results, due to the high cellular LASP-1 content of erythrocytes. In the examples described herein it has been found that only 19 % of patients with TCC exhibited a hematuria (i.e. is the presence of erythrocytes) of 100-200/ul of unprocessed urine The other patients with TCC exhibited no ria or a hematuria of below 100—200/ul of essed urine. According to current guidelines a hematuria is 35 preferably to be further investigated, by cystoscopy [1] .

WO 2013/083690 PCT/EP2012/074634 10 As described in the examples, urine samples may be controlled for erythrocytes with urine sticks (Combur 10 Test M, Roche, Mannheim, Germany). Urine samples comprising erythrocytes may ted to a cell-lysis step specifically Iysing erythrocytes. Alternatively erythrocytes may be d from a urine sample by magnetic beads specifically binding to surface marker of erythrocytes. Surface s for erythrocytes are well-known and include, for example, glycophorin A (CD235a).

According to a different preferred embodiment of the invention the transitional cell carcinoma is transitional cell carcinoma of the urine bladder, pelvis of the ureter, ureter, urethra, urachus, 10 kidney or ation thereof.

As It is detailed herein above the transitional cell carcinoma may affect one or more of these organs and organ appendages, 15 rmore, in accordance with another preferred embodiment of the invention the detection of LASP-1 comprises Western Blot analysis, mass spectrometry analysis, FACS—analysis, and ELISA.

Western Blot analysis, mass spectrometry analysis, FACS-analysis, and ELISA are non- 20 limiting examples of methods which may be used to qualitatively, semi-quantitatively and/or quantitatively detect LASP-1. In the examples herein below Western Blot analysis was used for the detection of LASP—1.

Western blot analysis is a widely used and well—know analytical que used to detect 25 specific proteins in a given sample, for e, a tissue homogenate or body extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The proteins are then transferred to a ne (typically ellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein. 30 Also mass ometry (MS) analysis is a widely used and now analytical technique, wherein the mass-to—charge ratio of charged particles is measured. Mass ometry is used for determining masses of particles, for determining the elemental composition of a sample or le, and for elucidating the chemical structures of molecules, such as 35 proteins, peptides and other chemical compounds. The MS principle ts of ionizing al compounds to generate charged molecules or molecule fragments and measuring WO 2013/083690 PCT/EP2012/074634 1 1 their mass-to-charge ratios.

Fluorescence activated cell sorting (FACS) analysis is a widely used and well—know analytical technique, wherein biological cells are sorted based upon the specific light scattering of the scent characteristics of each cell. Therefore, cells were fixed in 4 % formaldehyde, permeabilized with 0.2 % Triton-X400, and incubated with a fluorophore-labeled antibody (e.g. mono- or polyclonal LASP-1 antibody).

Enzyme—linked immunosorbent assay ) is a widely used and well-know sensitive 10 analytical technique, wherein an enzyme is linked to an antibody or antigen as a marker for the detection of a specific protein.

In accordance with a further preferred ment of the ion the LASP—1 runs as a 38 kDa protein in Western Blots. 15 Human LASP-1 is preferably a protein having 261 amino acids (most preferably the 261 amino acids of NCBI protein accession number NP_OO6139.1 which is shown in SEQ ID NO: 1). 20 According to a preferred embodiment the method of the invention r comprises (i) an tion of an endoscopy of the urinary bladder via the urethra in the subject from which the urine sample has been obtained, and/or (ii) a cytology examination of the urine sample for abnormal cells. 25 At t opy (i.e. endoscopy of the urinary bladder via the urethra) and urinary cytology (i.e. cytology examination of the urine sample for abnormal cells) are mainly used in order to diagnose and grade transitional cell carcinoma. Although both diagnostic methods suffer from the limitations discussed herein, they may be useful to ment the diagnosing and g of transitional cell carcinoma by using LASP-1 as defined herein above. 30 in another embodiment the invention relates to a kit for diagnosing and/or grading transitional cell carcinoma comprising (a) means for the detection and/or quantification of LASP-1 in a urine sample obtained from a subject and (b) ctions for using the kit. 35 With regard to the instructions of the kit it is preferred that it is described therein that a LASP- 1 level of above 1 ng/SOOuI of (unprocessed or processed) urine diagnoses a subject having a WO 2013/083690 2012/074634 12 tional cell carcinoma. It is also preferred with regard to the instructions of the kit that it is described therein that an increase of LASP-1 expression correlates with higher grading of the transitional cell carcinoma.

The ctions may furthermore comprise information on the method of the detection of LASP-1. For example, information may be provided on assays based on protein detection.

Such assays include t limitation enzyme-linked immunosorbent assay (ELISA), mass spectrometry, ion exchange chromatography, gel filtration chromatography, affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, disc 10 gel electrophoresis, Western blot analysis, immunoprecipitation, see, for example, Soejima and Koda, Transfusion 45 (2005) 1934—1939; Yeh et al., Anesth. Analg. 101 (2005) 1401- 1406; Chou et al., Am. J. Clin. Pathol.. 124 (2005) 330-338. As it described herein above in greater detail, the detection of LASP-1 may, for example, comprise Western Blot analysis, mass spectrometry analysis and/or FACS-analysis. 15 in accordance with the kit, “means for the detection and/or quantification of LASP—1” preferably comprise a “molecule binding to LASP-i” and more preferably a “molecule specifically binding to LASP-1”. Examples of les (specifically) binding to LASP-1 are bed herein above in more detail. 20 In ance with a preferred ment of the invention the means for the detection and/or quantification of LASP—1 is an antibody specifically binding to LASP—1 The s show: 25 Figure1 LASP-1 immunostaining in human bladder tissue. A and B: Normal bladder lium. C: Transitional cell carcinoma. Healthy tissue (thin arrows) and tumor (fat arrows).

D: Transitional cell carcinoma. E and F: LASP-1 positive ureter . G: LASP-1 positive tumor cells (arrows) from a patient with bladder cancer and (H) from a healthy eer in 30 cytospin specimen. All samples DAB, brown. Magnification 100x for A,C and E, all others 400x) Figure2 A: Western Blot example of LASP—1 is and calculation in control and patient urine samples. B: Western Blot analysis and calculation of LASP—1 levels in urine 35 samples contaminated with a fixed number of erythrocytes ul urine), respectively, red blood cells per high-power field (RBC/hpf). Shown are also the corresponding urine dipsticks WO 2013/083690 PCT/EP2012/074634 13 and the visible red blood cell pellets after sedimentation of the urine probes. Hematuria >200 Erys/ul urine are contraindicative.

Figure 3 u staining and LASP—1 Western Blot analysis of urinary pellets from control volunteers and TCC patients. LASP—1 protein concentrations are not necessarily related to l protein amount of the probes.

Figure 4 Receiver operating characteristic (ROC)—analysis of urinary LASP—1 content in bladder cancer. Area under curve was 70.0 % (95 % ence interval: O..544 to 0.854). 10 Figure5 A: LASP-1 knockdown inhibits cell migration. T24 bladder cancer cells transfected with LASP—1 siRNA and led control siRNA were seeded in modified Boyden chambers and incubated for 4 h. Migrating cells were fixed with paraformaldehyde and stained with crystal violet. The absorbance was measured. Bars, SEM (n=6); *** Students 15 ttest, p<0.0005; versus control. ments were performed three times with r results.

B: Increased adhesion in -depleted cells. T24 bladder cancer cells were seeded in 48 well plates coated with fibronectin and incubated for 4h. Cells were fixed with paraformaldehyde and stained with l violet. Absorbance was measured at 595 nm.

Bars, SEM (n=8); ** Students ttest; p<0.001, versus control. C: LASP—1 knockdown does not 20 influence cell proliferation. LASP—1 siRNA-transfected T24 cells were seeded in 48 wells, incubated with MTT/dye solution for 4 h and the absorbance was recorded. Bars, SEM. ments were performed three times with similar results.

The examples illustrate the invention. For all experiments, LASP-1 knockdown efficiency was 25 controlled by Western blots.

Example 1 - Materials and Methods Tissue collection and immunohistochemistry 30 ts background and clinicopathologic characteristics are summarized in Table 1. 35 WO 2013/083690 PCT/EP2012/074634 14 Table 1: Patients characteristics —-_ The samples were staged according to the Union Internationale Contre le Cancer (UICC).

Paraffin-embedded tissue samples of 72 ed human urinary bladder with confirmed histological diagnoses and 17 incidental healthy ureter samples from patients undergoing nephrectomy for renal cell cancer were obtained from the ment of Pathology, University of urg. 10 histochemistry For immunohistochemical staining procedures two tissue sections were cut from each regular paraffin ed tissue at 2—3 um. For immunostaining, sections were placed onto APES (3— amino-propyltriethoxy—silane; Roth, Karlsruhe, Germany) coated slides, dewaxed in xylene, rehydrated in graded ethanol and in TRIS—buffered saline (TBS; 25 mM TRlS/HCi, pH 7.4, 137 15 mM NaCl, 2.7 mM KCl). For antigen retrieval, sections were subjected to heat pretreatment by boiling it in 0.01 M of sodium citrate buffer (pH 6.0) for 10 min in a microwave oven (600Watt/sec.). Endogenous peroxidase was blocked by incubation in 0.1% hydrogen peroxide in PBS for 5 min. Slides were then incubated with the poiyclonal anti-LASP-1 antibody [12] diluted 1:1000 in ody diluent” (DAKO, Hamburg, y) followed by EnVlsion/rabbit 20 detection system (DAKO, Hamburg, Germany). For Hematoxylin (HE)—staining, 3,3’- Diaminobenzidine (DAB; DAKO, Hamburg, Germany) was used as chromogen and sections were rstained in hematoxylin (Mayers, Sigma, Deisenhofen, Germany), dehydrated through graded ethanol and embedded in Entelan (Merck, Darmstadt, Germany). All HE samples WO 83690 PCT/EP2012/074634 15 were examined by an expert pathologist to confirm the previous diagnosis. All immunohistological samples were evaluated by the pathologist (A.S.) and an ndent observer for defining of the percentage of LASP-1 positive cells and the staining intensity.

Scoring of cytosolic LASP—1 expression was d out in analogy to scoring of hormone receptor Immune Reactive Score (IRS), ranging from 0—12 according to Remmele et al. and is described in detail for LASP—1 in breast cancer cells [9, 13].

Patient population for urine analysis In total 48 healthy volunteers and 84 ts undergoing either tomy for verified 10 muscle ve TCC or transurethral resection for ted bladder tumor in the two major urological urological deparments in Wuerzburg and Freiburg were included. LASP—1 content of urinary cell pellet was ed in 132 cases and urine dipsticks analyses performed in parallel. Exclusion criteria were gross hematuria and urinary infection. Approvals of the local ethic committees in Freiburg and Wuerzburg were ed. All participants gave written 15 informed consent.

Western blot analysis for urinary LASP—1 From all urine s, 10ml of urine was centrifuged immediately at 3.3009 for 10min at RT in a 15 ml tube. Independent of quantity, each pellet was resuspended in 200 pl Laemmli 2O sample buffer, heated at 95°C for 5min and stored at -20°C for further processing. Of each sample, 10 ul (corresponding to 500 pl urine) were resolved by 10% SDS—PAGE. After blotting on nitrocellulose membrane and blocking with 3% nonfat dry milk in 10 mM Tris, pH 7.5, 100 mM NaCl, 0.1% (w/v) Tween 20, the membrane was first incubated with the antibody raised against LASP-1 00) [12] followed by incubation with horseradish peroxidase- 25 coupled goat anti-rabbit IgG (Biorad, Munich, Germany), diluted 1:5000, and visualization was done using ECL (GE Healthcare, rg, Germany). Protein bands were visualized by autoradiography. Quantification of autoradiography signals was carried out by densitometry using the lmageJ software (NIH, Bethesda, USA). 30 To ine release of LASP—1 from urothelial cells into the urine, 200 pl supernatant samples were processed in parallel for Western blot analysis.

To analyse the influence of hematuria two healthy donor urine samples were supplemented with increasing amounts of fresh blood imitating erythrocytes contamination in the range of 35 10-500 cells / pl urine. Samples were controlled with urine sticks (Combur 10 Test M, Roche, Mannheim, y) and then processed as described above. Recombinant human LASP-1 WO 2013/083690 PCT/EP2012/074634 16 served as standard concentration [12].

Statistical analysis Sensitivity, specificity, as well as positive and negative predictive values of urinary LASP-1 levels were investigated after setting a cut off level by ROC-analysis. Confidence interval was 95%. All analysis were performed with GraphPad Prism 5.

Cell line and culture ions The human bladder carcinoma cell line T24 (purchased from ATCC) was grown at 37°C 10 under 5% C02 atmosphere in McCoy medium (lnvitrogen, Karlsruhe, Germany) containing 10% heat-inactivated fetal bovine serum (PAA, Linz, Austria) and 1% streptomycin/ampicillin (lnvitrogen, Karlsruhe, Germany).

Suppression of LASP-1 15 LASP1 down was performed using two siRNA constructs targeting the LASP1 sequences 5’-AAG GTG AAC TGT CTG GAT AAG—3’ (bases 49-69) and 5'-AAG CAT GCT TCC ATT GCG AGA ~3‘ (bases 80-100); (ordered from Dharmacon, tte, CO). Both siRNA exhibited LASP-1 own of 75-80 %. Non-targeting siRNA #5 from con was used as scrambled control. Cells were plated at a density of 2 x 105 cells/ 25 cm2 flask, 20 grown for 24 h at a confluence of 30—50% and transfected with 30 ul siRNA stock solution mixed with 30 ul HiPerfect (Qiagen, Hilden, Germany) in 100 pl reduced serum medium OPTI-MEM-l (Gibco, Paisley, UK) according to the manufacturers protocol. After 4 h incubation at 37°C, transfection medium was replaced by 5 ml routine cell culture medium and incubation was continued for 42 h. For scrambled control cells, 30 ul non-targeting siRNA (20 25 uM) was used. For adhesion and ion ments, the cells were synchronized by starving overnight in basal medium with 0.5% FCS. At least three independent experiments were performed for each cell line, and representative s are shown. LASP1 knockdown was controlled by Western blots in all ments. 30 Adhesion, proliferation and migration experiments To assess cell adhesion, 48-well plates were coated with 10 ug/ml fibrinogen (Sigma, Deisenhofen, Germany) diluted in PBS and 0.1% BSA ght at 4°C. Cells were washed in serum—free medium, re-suspended at a concentration of 5 x 105 cells/ml and 200 pl were seeded. Cells were allowed to attach for 4 h at 37°C. Non-adherent cells were removed by 35 gentle washing with PBS. Attached cells were fixed in 4% (w/v) paraformaldehyde for 10 min and then stained with 0.5% (w/v) crystal violet (in 2% l, filtered with 0.45 uM pore size) WO 2013/083690 PCT/EP2012/074634 17 for 20 min followed by 3 times washing with PBS. The blue dye was eluted in 10% acetic acid for 10 min, and the absorbance was measured at 595 nm on a plate . Adhesion assays were performed in 3 independent experiments with both LASP—1 siRNAs, each with 6 replicates Proliferation was determined by the MTT-based ter96 AQ dioactive Cell Proliferation Assay (Promega, Mannheim, y). in brief, cells were seeded at 1 x 104 cells/48—well and cultured overnight at 37°C. After 24 h, cells were transfected with LASP1— specific siRNA and cultured for r 48 h. Transfection reagent and scrambled siRNA 10 controls were included in all ments. After the incubation period, 30 pl MTT dye solution was added. After an incubation of 4 h at 37°C, 200 pl STOP-solution (0.1 N HCl, 10% SDS) was added and the plates were incubated at RT for another hour. Cells were solubilized by pipetting, transferred to a 96-well plate and the absorbance was recorded using a plate reader. Cell proliferation was sed as percentage of control cells. Experiments were 15 done twice with both LASP-1 siRNAs in 8 replicates using separate cell cultures.

The migration assay was performed using a modified Boyden chamber assay (Transwell chambers, Corning Star, dge, MA). in brief, cells were starved overnight, trypsinized, adjusted for viability, counted and re-suspended in serum-free medium with 1 mM 20 MgCl2 to a concentration of 1 x 106 cells/ml. The lower surface of the filter membrane (8 pM pore size) was yed with 100 pl fibronectin on (5 ug/ml; Sigma, Deisenhofen, Germany) as a chemoattractant for 30 min before adding 100 pl cell suspension into the BSA- coated filter chamber. The filter chambers were cultured in 500 pl routine medium with 10% FCS for 4 h at 37°C to allow the cells to migrate through the porous membrane. Cells 25 remaining at the upper surface were completely removed using a cotton swab. Cells at the lower surfaces of the membranes were stained in a solution of 1% (w/v) crystal violet in 2% ethanol for 30 s and rinsed afterwards in distilled water. Cell—associated crystal violet was extracted by incubating the membrane in 200 pl 10% acetic acid for 20 min and measured at 595 nm absorbance using a 96-well plate reader. Experiments were done twice in 30 quadruplicate with both LASP-1 siRNAs.

Example 2 - LASP-1 expression is ubiquitous and moderately elevated in TCC in immunohistochemistry 35 The immunohistochemical distribution of LASP-1 in bladder cancer specimen of 72 patients undergoing either transurethral ion or cystectomy was investigated.

WO 2013/083690 PCT/EP2012/074634 18 Only 6 samples out of these 72 TCC were attested to be LASP—1 negative determined by the intensity of the staining. All other tumors were stated LASP—1 positive. A consistant basal level of LASP-1 expression was noted in the nding y urothelium (Figures 1A-D). in order to e urothelium without a le precancerous influence healthy urothelium from incidental ureter s and observed a consistent LASP-1 expression throughout the urothelium was stained (Figures 1E and 1F). Analysis of the Immune Reactive Score showed that expression in TCC was higher than in the surrounding tissue or healthy tissue of the ureter but differences were only moderate, regardless of tumor cell grading. 10 Example 3 - Urinary LASP-1 is a sensitive marker for TCC in order to investigate the presence of LASP-1 in voided urine of bladder cancer patients and to establish the diagnostic / prognostic possibilities of this marker protein, a total of 132 15 y sediments for the presence of LASP-1 by Western blot analysis was analysed. Pellets corresponding to 500 pl urine were ted by SDS—gel and LASP-1 concentration was determined according to the standard by densitometry. The cut—off point was set to 1ng LASP-1 / 500 pl urine by ROC analysis, see below. In Figure 2A a representative Western blot with LASP-1 standard, control samples and patient s is shown. While only control 20 sample No. 40 is false positive, all patients exhibit a detectable LASP—1 band > 1ng / 500 pl urine at 38 kDa. LASP-1 protein levels are not necessarily related to overall protein amount of the probes (Figure 3).

It should be noted that urine cytology (cytospin) as well as exfoliative bladder wash did not 25 further enhance the diagnostic specificity of LASP-1 for TCC mostly due to insufficient cell counts but also because of only moderate differences in LASP-1 staining between healthy and tumor cells (Figure 1G and 1H).

To assess whether soluble LASP—1 is ed into the urine of bladder cancer patients, 30 onal Western blot analysis with the urine supernatant from 3 patients with low (0.18 ng / 500 pl urine), medium (1.8 ng / 500 pl urine) and high (7.8 ng / 500 pl urine) LASP—1 levels in the urine sediment was performed. Soluble LASP-1 was not detected in any of the urinary supernatant probes (data not shown). 35 As microscopic hematuria is a common ion in bladder cancer it was sought to assess cross-reactivity of the LASP-1 antibody with contaminated protein from blood cells in the WO 2013/083690 2012/074634 19 urinary sediment. y-voided urine from two healthy volunteers without any medications and added blood to the urine to give known concentrations of hematuria was used. Urine was controlled by dipsticks and high-power field microscopy (hpf). As seen in Figure 28, blood up to 100 erythrocytes / pl urine (5-10 RBC/hpf) is by tion negative for LASP-1 (< 1ng) in the Western blot. Only after a red blood cell concentration beyond 200 erythrocytes / pl urine a weak LASP-1 band (> 1ng) was ed and a clearly visible red pellet could be observed in the urine sediment (Figure 28).

To determine the ideal cut off point for urine LASP-1 content a ROC analysis was med 10 on the results of the first 52 samples and determined an ideal cut off point of 1ng LASP- 1/500pl (Figure 4). Area under curve was 70.0% (95% ence interval: 0.544 to 0.8537).

Using this value, sensitivity and specificity for detection of TCC was 83.1% and 85.3%, respectively. Positive and negative predictive value was 83.1% and 80.6%, respectively (Table 2). Sensitivity for low vs high grade tumors was 65% and 87%. Sensitivity for non- 15 muscle invasive tumors was 74% and for muscle invasive tumors 94.1%. A correlation analysis of LASP-1 t with grading revealed a clear increase of LASP—1 expression with higher grading (data not shown). In hematuria ve ts, sensitivity and icity were 79.2 and 84.8%, respectively. 20 Table 2: Diagnostic value of LASP-1 measurement in urine sediment Sensitivity 33.1 % Specificity 883 9’.

Positive pmdlctive value 8211 83 Negative predictive value 30.3 #3 Example 4 - In vitro inhibition of LASP-1 attenuates the malignant phenotype of bladder 25 cancer cells and reduces cell detachment Cell migration, adhesion and proliferation was investigated in the human bladder cancer cell line T24 to investigate the reason for the increased LASP-1 content in urinary cell pellets. siRNA—mediated knockdown caused profound reduction of LASP-1 protein abundance in the 30 cell line and was confirmed by Western Blot with maximum knock down 2 80% detected after WO 83690 PCT/EP2012/074634 20 48 hours (Figure 5).

By using a modified Boyden-chamber and a fibronectin on assay, a reduction in migratory potential (Figure 5A) accompanied by an enhanced cell adhesion in T24 cells upon LASP-1 knockdown was observed e SB), whereas no significant inhibition of proliferation was observed by the MTT assay in the cells upon LASP-1 knock down e 50). ture 10 1. Babjuk M, Oosterlinck W, Sylvester R, et al: EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder, the 2011 update. European urology 59:997-1008 2. Cooksley CD, Avritscher EB, Grossman HB, et al: Clinical model of cost of bladder cancer in the elderly. Urology 71:519-525, 2008 3. ti S, Hu J, Pearce I: Patients' acceptance of repeated invasive ical 15 igations. BJU international 103:1453-1454, 2009 4. Tilki D, Burger M, Dalbagni G, et al: Urine Markers for Detection and llance of Non-Muscle-lnvasive Bladder Cancer. European urology 5. Grunewald TG, Butt E: The LIM and SH3 domain protein family: structural proteins or signal transducers or both? Molecular cancer 7:31, 2008 20 6. Chiyomaru T, Enokida H, Kawakami K, et al: Functional role of LASP1 in cell viability and its regulation by microRNAs in bladder cancer. Urologic oncology 7. Pappas CT, Bliss KT, Zieseniss A, et al: The n family: an actin support group.

Trends in cell biology-21:29-37 8. Frietsch JJ, Grunewald TG, Jasper S, et al: Nuclear localisation of LASP-1 correlates 25 with poor long-term survival in female breast cancer. British journal of cancer 102:1645-1653 9. Grunewald TG, Kammerer U, Kapp M, et al: Nuclear localization and lic overexpression of LASP—1 correlates with tumor size and nodal-positivity of human breast carcinoma. BMC cancer 7:198, 2007 10. Zhao L, Wang H, Liu C, et al: Promotion of colorectal cancer growth and metastasis by 30 the LIM and SH3 domain protein 1. Gut 59:1226—1235 11. Chew CS, Parente JA, Jr., Zhou C, et al: Lasp-1 is a regulated phosphoprotein within the cAMP ing pathway in the gastric parietal cell. The American journal of physiology 275:056-67, 1998 12. Butt E, Gambaryan S, Gottfert N, et al: Actin binding of human LIM and 8H3 protein is 35 regulated by cGMP- and CAMP-dependent protein kinase orylation on serine 146. The Journal of biological chemistry 278:15601-15607, 2003 WO 2013/083690 PCT/EP2012/074634 21 13. Remmele W, Stegner HE: [Recommendation for uniform definition of an immunoreactive score (lRS) for immunohistochemical estrogen receptor detection (ER-lCA) in breast cancer tissue]. Der Pathologe 8:138-140, 1987 14. Traenka C, Remke M, Korshunov A, et al: Role of LIM and 8H3 protein 1 (LASP1) in the metastatic dissemination of medulloblastoma. Cancer ch 70:8003-8014 15. Patriarca C, Colombo P, Pio Taronna A, et al: Cell discohesion and multifocality of oma in situ of the bladder: new insight from the adhesion molecule profile herin, Ep-CAM, and MUC1). international journal of surgical pathology 17:99-106, 2009 16. Yao R, Lemon WJ, Wang Y, et al: Altered gene expression profile in mouse bladder 10 s induced by hydroxybutyl(butyl)nitrosamine. sia (New York, NY 6:569—577, 2004 17. Grunewald TG, Kammerer U, Winkler C, et al: pression of LASP-1 mediates migration and proliferation of human ovarian cancer cells and influences zyxin localisation.

British journal of cancer 96:296—305, 2007 15 18. Mihlan S, Reils C, Thalheimer P, Herterich S, Gaetzner S, skothen J, Pavenstz'a'dt HJ, Lewandrowski U, nn A, Butt E. Nuclear import of LASP-1 is regulated by phosphorylation and dynamic protein-protein interactions. Oncogene. 2012 Jun 4. doi: 10.1038/onc.2012.216. [Epub ahead of print] PubMed PMID: 22665060.

Claims (13)

1. Use of LASP-1 in a urine sample obtained from a subject for diagnosing and/or grading transitional cell carcinoma, wherein a LASP-1 level of above 1 ng/500μl 5 of urine diagnoses a subject as having transitional cell carcinoma, and an increase of LASP-1 expression correlates with a higher grading of the transitional cell carcinoma.

2. A method for sing transitional cell carcinoma comprising detecting the presence or absence of LASP-1 in a urine sample obtained from a subject, wherein the presence of LASP-1 above 1 ng/500µl urine is indicative for transitional cell carcinoma. 10

3. A method for grading tional cell carcinoma comprising determining the level of LASP-1 in a urine sample obtained from a subject, wherein the level of LASP-1 correlates with the grade of the transitional cell carcinoma.

4. The method of claim 2 or 3, wherein the urine sample is urine, a y cell pellet or a resuspended urinary cell pellet. 15

5. The method of any one of claims 2 to 4, wherein the urine sample has been obtained from a subject not having a urinary tract infection.

6. The method of any one of claims 2 to 5, wherein the urine sample comprises less than 250 erythrocytes per µl urine, ably less than 200 erythrocytes per µl urine and more preferably less than 150 erythrocytes per µl urine. 20

7. The method of any one of claims 2 to 6, wherein the transitional cell carcinoma is transitional cell carcinoma of the urine bladder, pelvis of the ureter, urethra, urachus, kidney or combination thereof.

8. The method of any one of claims 2 to 7, wherein detecting LASP-1 comprises n Blot analysis, mass spectrometry analysis, FACS-analysis or ELISA. 25

9. The method of any one of claims 2 to 8, n the LASP-1 is a 261 amino acid protein that runs as a 38 kDa protein in n blots.

10. The method of any one of claims 2 to 9, further comprising (i) the evaluation of the results of an endoscopy of the urinary bladder via the urethra in the subject from which the urine sample has been obtained, and/or (ii) a cytology ation of the urine sample for abnormal cells. 5

11. Use according to claim 1, substantially as herein described with reference to any one of the examples but excluding comparative examples.

12. A method ing to claim 2, substantially as herein described with reference to any one of the examples but excluding comparative es.

13. A method according to claim 3, substantially as herein described with 10 reference to any one of the examples but excluding comparative examples.