Patent Application: US-201113808000-A

Abstract:
the invention refers to a novel molecular biomarker , namely ptpd1 , that is markedly increased in human bladder cancers . ptpd1 expression positively correlated with the grading and invasiveness potential of these tumors . ptpd1 can be detected at high levels in exfoliated bladder cells isolated from urine of bladder cancer patients , while no ptpd1 signal was evident in normal exfoliated bladder cells . thus , ptpd1 detection in urine samples may represent a novel and reliable marker for non - invasive diagnosis of aggressive bladder cancer .

Description:
1 . ptpd1 is overexpressed in human bladder cancer tissues . ptpd1 has been implicated in critical aspects of cell - cell communication , growth and motility . this led us to investigate the expression profile of ptpd1 in human bladder epithelial cancer with high recurrence rate and metastatic behavior . the risk grade of cancer lesions was assessed basing upon the categorization provided by the who bladder grading . tissues samples were derived from first biopsies of patients affected by urothelium hyperplasia , urothelial papilloma , low malignant potential urothelial neoplasia , low grade or high grade urothelial carcinoma . by immunoblot analysis and immunohistochemistry , authors found that ptpd1 is nearly undetectable in normal tissue , in hyperplastic urothelium and urothelial papilloma , while low - moderate levels are present in low - grade urothelial carcinoma . in contrast , ptpd1 strongly accumulated in samples derived from high - grade urothelial carcinomas . the analysis was carried out on a total of 46 patients : 12 normal or hyperplastic urothelium , 3 urothelial papilloma , 15 low grade urothelial carcinoma and 19 cases of high grade urothelial carcinoma . data on ptpd1 overexpression in bladder cancer specimens was confirmed in a large number ( about 500 ) of human bladder cancers by tissue microarray analysis ( tma ). 2 . ptpd1 is detected of in exfoliated bladder cancer cells . authors analyzed ptpd1 levels in exfoliated bladder cells recovered from urine samples . cells from urine specimens of control and bladder cancer patients were subjected to immunocytochemistry using anti - ptpd1 antibody generated by authors using standard techniques . the data demonstrate that ptpd1 was nearly undetectable in exfoliated , normal bladder cells from healthy volunteers . in contrast , urine cancer cells from bladder patients show a strong ptpd1 immunoreactive signal , compared to normal cells present in the same urine specimen where ptpd1 staining was undetectable . thus , ptpd1 detection in urine samples represents a novel and reliable marker for non - invasive diagnosis of aggressive bladder cancer . ptpd1 detection may be achieved with techniques known to the skilled person , as immunodetection of the protein itself or of fragments thereof , enzymatic assays , ptpd1 rna detection . it is therefore an object of the invention a method for the diagnosis or the monitoring control for therapy of bladder cancer characterized in detecting the ptpd1 protein or an immunological fragment thereof , or the ptpd1 enzymatic activity , or the ptpd1 mrna in a body sample . the ptpd1 protein is a protein having essentially the sequence of seq id no . 1 or an allelic variant thereof . preferably the detecting of the ptpd1 protein or of an immunological fragment thereof is performed by :— allowing the body sample to react with a ptpd1 protein specific ligand to form a complex ;— detecting the complex . more preferably said specific ligand is an anti - ptpd1 antibody ( monoclonal or polyclonal ) or an immunological , synthetic or recombinant derivative thereof . the anti - ptpd1 antibody or immunological , synthetic or recombinant derivative thereof is obtainable by using as immunogen the whole ptpd1 protein of seq id no . 1 or an immunogenic fragment thereof . preferably the immunogenic fragment of the ptpd1 protein of seq id no . 1 is comprised between aa 751 and aa 910 of seq id no . 1 . alternatively the immunogenic fragment of the ptpd1 protein of seq id no . 1 consists of a sequence between aa 751 and aa 910 of seq id no . 1 . in a preferred embodiment the detecting step is by means of detection of a specific fluorescent signal . in an alternative preferred embodiment of the method of the invention the detecting of the ptpd1 enzymatic activity is performed by fluorescent or radiolabeled assays , i . e . based on ptpd1 - catalyzed release of the phosphate group from a given substrate . in an alternative preferred embodiment of the method of the invention the detecting of the ptpd1 mrna is performed by northern blot analysis , polymerase chain reaction ( pcr ) or pcr - derived methods , or nucleic acid amplification based methods . the method of the invention is performed on a body sample out of the body , as a bladder tissue , or body fluid or a fraction thereof , i . e . urine or its sediment . it is a further object of the invention a bladder cancer diagnostic kit comprising : a solid phase adhered ptpd1 protein specific ligand as above disclosed ; a detection ligand able to bind to ptpd1 protein specific ligand — ptpd1 protein . preferably the ptpd1 protein specific ligand is an anti - ptpd1 antibody or a derivative thereof , as above disclosed , more preferably said anti - ptpd1 antibody or derivative thereof is obtained by using a fragment of the ptpd1 protein as antigen , more preferably in the region between aa . 618 and aa . 910 of the ptpd1 protein . fig1 . a . schematic representation of the human ptpd1 protein . ptp , catalytic domain ; acr , acidic region ; ferm , four point one - ezrin - radixin - moesin domain binding domains for src , actin and fak are indicated . fig2 . ptpd1 is highly expressed in bladder carcinomas . a - b . tumor samples ( t ) were isolated from patients affected by high grade ( lanes # 2 , # 3 , # 4 , # 6 , # 7 , # 8 ) or low grade ( lanes # 1 , # 5 , # 9 ) urothelial carcinoma . normal tissue ( n ) surrounding each neoplastic lesion was also isolated . tissue samples were lysed , resolved on 8 % sds - page gels and immunoblotted with the following antibody : anti - peptide ptpd1 ( ab1 ) ( a ) or anti - polypeptide ptpd1 ( ab2 ) ( b ), anti - erk2 and anti - cytokeratins . c . tissue sections from normal bladder ( a ), hyperplastic bladder ( b and c ) and high grade ( d ) of urothelial carcinoma were immunostained with anti - ptpd1 antibody and analyzed by light microscopy . higher resolution panels ( a ′, b ′, c ′, d ′) of each set of images are shown on the right . d . bladder lesions were subgrouped in three categories : a . normal / hyperplastic ; b . low grade urothelial carcinoma ; c . high grade urothelial carcinoma . cumulative data and relative abundance of ptpd1 in each category are shown . fig3 . tissue microarray analysis ( tma ) for ptpd1 expression in human bladder biopsies . tissue microarray of 505 bladder samples ranging from normal tissue , benign lesions and urothelial carcinomas was immunostained with anti - ptpd1 polyclonal antibody . a . enlarged section of representative biopsies of normal and cancer lesions immunostained with anti - ptpd1 antibody . b . cumulative data are expressed as percentage of ptpd1 - positive samples within the two main categories ( normal / hyperplastic lesions and urothelial carcinomas ). p value is indicated on the right column c . ptpd1 - positive urothelial carcinomas were scored for ki - 67 positivity . cut - off value represents the percentage of ki - 67 positive cells versus total cells scored . d . inverse correlation between bladder stage disease ( pta , pt1 and pt3 ) and ptpd1 signal . the analysis was carried out on a total of 349 patients with urothelial carcinoma . fig4 . ptpd1 is present in urinary exfoliated bladder cells . urine samples from control ( a ) and bladder cancer patient ( b ) were subjected to immunocytochemistry using anti - ptpd1 antibody . a representative experiment of ptpd1 detection in urine samples from bladder cancer patients (# 5 ) is shown . enlarged section of the panel b is also shown ( b ′). arrows indicate normal and urothelial cancer cells . human bladder samples . tissue samples were isolated from patients affected by benign and malignant tumors of the urinary bladder , along with patients affected by hyperplastic or normal urothelial mucosa were retrieved from the files of the department of biomorphological and functional sciences , pathology section , and department of urology , university “ federico ii ” of naples , italy . the risk grade was assessed basing upon the categorization provided by the who bladder grading 2004 . anti - ptpd1 polyclonal antibodies , anti - ptpd1 polyclonal antibodies were generated as follows as already disclosed ( carlucci et al . 2008 ). a cdna encoding for the central core of human ptpd1 protein ( aa . 751 - 910 ) was subcloned in prset - vector , expressed in bl21 bacteria and affinity purified on column the purified fragment was used to immunize rabbits . the specificity of the antibody was tested by western blot and immunofluorescence ( carlucci et al ., 2008 ). immune anti - ptpd1 igg were also produced . immunohistochemistry . formalin - fixed , paraffin embedded tissues from patients subjected to cystoscopic biopsy were selected . representative slides of each tumor were stained with hematoxylin and eosin to confirm the diagnosis and grading of the tumors . 4 - μm serial sections from representative blocks were cut , mounted on poly - l - lysine coated glass slides and used for the immunohistochemical examination . two polyclonal antibodies raised against distinct domains ( residues 751 - 910 ; residues 618 - 631 ) of human ptpd1 protein ( seq id no . 1 ) were used . the antibody raised against residues 618 - 631 was previously described ( moller et al ., 1994 ). both anti - ptpd1 antibodies gave identical immunostaining pattern . sections were de - paraffined in xylene and rehydrated through a decreasing concentration of alcohol to water . before incubation with the antibodies the slides were heated in a pression cooker for 3 minutes in a solution of 0 . 01 mol / l sodium citrate ( ph 6 . 0 ). to avoid non - specific binding , sections were pre - incubated with non - immune serum ( 1 : 20 , dakopatts , hamburg , germany ) diluted in pbs / bsa , 1 %, for 25 minutes , at room temperature . endogenous peroxidases activity was reduced by incubation with 3 % hydrogen peroxide for 20 minutes . representative sections were incubated with the listed primary antibodies , overnight at 4 ° c . subsequently , the slides were incubated with biotinylated secondary antibodies , peroxidase - labelled streptavidin ( dako lsab kit hrp , carpinteria , calif .) and chromogenic substrate diaminobenzidine ( dab , vector laboratories , burlingame , u . s . a .) for the development of the peroxidase activity . slides were counterstained with hematoxylin , dehydrated and cover - slipped with a synthetic mounting medium ( entellan , merck , germany ). omission of primary antibody and substitution with phosphate - buffered saline were used as negative controls . section analysis was performed by two pathologists blind to the histological typing and to the follow - up data of the single cases of bladder carcinoma . only cells with a definite membrane and cytoplasmic staining were judged as positive for each antibody . ptpd1 is over - expressed in urothelial carcinomas . authors investigated the expression profile of ptpd1 in human bladder urothelial cancers , comparing tumors with different recurrence rates and metastatic potential . the risk grade of cancer lesions was based upon the categorization provided by the who bladder grading . tissues samples were derived from first biopsies of patients with urothelial hyperplasia , urothelial papilloma and low - or high - grade urothelial carcinoma . tissue samples were homogenized , and protein lysates were immunoblotted with anti - ptpd1 antibody . fig2 a shows that ptpd1 was nearly undetectable in normal bladder tissue , hyperplastic urothelium and urothelial papilloma , whereas low levels were visible in low - grade urothelial carcinoma . however , elevated ptpd1 concentrations were seen in samples derived from high - grade urothelial carcinomas . these tumors express high levels of cytokeratins , which are typical molecular markers of epithelial bladder cancer ( sanchez - carbayo et al ., 2006a ; sanchez - carbayo et al ., 2006b ) ( fig2 b ). similar findings were obtained using an anti - ptpd1 antibody raised against the aa 618 - 631 aa epitope peptide of seq id no . 1 , as previously described ( moller et al ., 1994 ). to address further this issue , authors performed immunohistochemistry on bladder sections derived from the same patients described in fig1 . the results shown in fig2 c are consistent with the immunoblotting data . ptpd1 accumulated in high - grade urothelial carcinoma , whereas ptpd1 levels were low - to nearly absent - in other tissue samples ( normal urothelium , urothelial hyperplasia and urothelial papilloma ). the results of this analysis , which was performed on a total of 46 patients , are summarized in fig2 d . authors also evaluated ptpd1 expression in a large number of human bladder cancers by tissue microarray analysis ( tma ). the array contained about 500 bladder samples ranging from normal tissue , benign lesions and urothelial carcinomas . tissue sections were subjected to immunohistochemistry using two different anti - ptpd1 antibodies that gave similar results . fig3 a shows over - expression of ptpd1 in two representative urothelial carcinomas , compared to normal bladder tissues . quantitative analysis ( fig3 b ) shows that ptpd1 was over - expressed in about 31 % of bladder carcinomas , whereas a low or undetectable immunoreactive signal was obtained in other samples , including normal or hyperplastic urothelium . ki - 67 is a proliferative marker and its cutoff value of 10 % is commonly used as predictive parameter of bladder cancer recurrence and progression ( blanchet et al ., 2001 ; liedberg et al ., 2008 ). as shown in fig3 c , a greater number of ptpd1 - positive sections were evident in malignant lesions with a ki - 67 cutoff value & gt ; 10 %, compared to those lesions with a ki - 67 value & lt ; 10 ( 64 % versus 36 %, respectively ). next , authors assessed whether ptpd1 immunoreactivity correlates with the staging of bladder disease . as shown in fig3 d , urothelial bladder cancers in an early developmental stage ( pta ) include more ptpd1 - positive cells ( 60 %) compared to cancers in intermediate ( pt1 ) ( 35 %) or advanced ( pt3 ) ( 23 %) disease stages . the inverse correlation between ptpd1 expression and disease progression might reflect a requirement of ptpd1 in an early step of tumor progression when cells first acquire a high proliferative rate and a more invasive behavior . next , authors sought to analyze ptpd1 levels in exfoliated bladder cells . cells from urine specimens of control and bladder cancer patients were subjected to immunocytochemistry using anti - ptpd1 antibody . a representative experiment of ptpd1 detection in urine samples from bladder cancer patients is shown in fig4 . ptpd1 was nearly undetectable in exfoliated , normal bladder cells from healthy volunteers ( fig4 a ). in contrast , urine cancer cells from bladder patients show a strong ptpd1 immunoreactive signal , compared to normal cells present in the same urine specimen where ptpd1 staining was undetectable ( fig4 b ). thus , ptpd1 detection in urine samples represents a novel and reliable marker for non - invasive diagnosis of aggressive bladder cancer . cardone , l ., carlucci , a ., affaitati , a ., livigni , a ., decristofaro , t ., garbi , c ., varrone , s ., ullrich , a ., gottesman , m . e ., avvedimento , e . v ., and feliciello , a . 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( 1994 ). src kinase associates with a member of a distinct subfamily of protein - tyrosine phosphatases containing an ezrin - like domain . proc natl acad sci u s a 91 , 7477 - 7481 . sanchez - carbayo , m ., socci , n . d ., lozano , j ., saint , f ., and cordon - cardo , c . ( 2006a ). defining molecular profiles of poor outcome in patients with invasive bladder cancer using oligonucleotide microarrays . j clin oncol 24 , 778 - 789 . sanchez - carbayo , m ., socci , n . d ., lozano , j . j ., haab , b . b ., and cordon - cardo , c . ( 2006b ). profiling bladder cancer using targeted antibody arrays . am j pathol 168 , 93 - 103 .