Patent Application: US-201214115259-A

Abstract:
the identification is disclosed of a new molecular factor for the early diagnosis of urothelial carcinoma of the bladder . htra1 is a molecule that is produced mainly by the urothelium of the bladder , both in physiological and inflammatory pathologies , such as cystitis , whereas it is absent in the urothelial layer of the neoplastic bladder mucosa , regardless of cancer grade and level . by means of western blotting two forms of htra1 were identified , both in bladder tissue and urine , the native 50 kda form and the autocatalytic 38 kda form . the 38 kda form is significantly underexpressed in tumoral tissues compared with normal tissues and concentration of the 50 kda form in urine is significantly higher in patients affected by bladder cancer compared to healthy subjects and subjects with cystitis . therefore , the evaluation of tissutal and urinary htra1 levels can be a specific marker of urothelial carcinoma of the bladder .

Description:
the procedure followed to achieve the aforesaid results is described hereinafter in detail . tissue samples of urothelial carcinoma with different malignity level ( 2004 who classification ) ( 30 ) and at different infiltration stages ( 2002 tnm classification ) ( 31 ) were collected . invasive tumoral tissues and visually normal tissue samples of the same bladder , far from the area affected by the pathology , were obtained from patients who underwent radical cystectomy ( n = 18 ), ( table 1 and 2 below ). moreover , samples of papuliferous tumoral tissue were taken with vesical biopsy from patients who underwent turb ( w = 30 ), as well as samples of inflamed tissue from subjects affected by bacterial cystitis ( n = 10 ) ( tables 1 , 2 ). patients affected by urothelial carcinoma and cystitis ( 68 . 2 average age ) also provided the urine samples used in this study . moreover , urine samples from healthy subjects ( n = 15 ) ( 62 average age ) were analyzed ( table 2 ). all patients , admitted at the urological clinic of the polytechnic university of marche , signed the informed consent and were not affected by any other pathology . step 2 — immunohistochemistry of samples of normal bladder tissue , tumoral bladder tissue and tissue from subjects affected by cystitis . for the immunohistochemical analysis ( table 2 ), the samples of normal bladder tissue , tumoral bladder tissue and tissue from subjects affected by cystitis were fixed in formalin at 4 %, included in paraffin and cut in 3 μm sections . the paraffin sections were deparaffined and rehydrated through passages in xylene and in a graduated series of alcohols with decreasing percentage ( from 100 % to 50 %), incubated for one hour with hydrogen peroxide at 3 % in deionized water to inhibit the activity of endogenous peroxidase and finally treated for 10 minutes at 37 ° c . with 0 . 1 % trypsin and 0 . 1 % calcium chloride in tris - hcl 0 . 05m ph 7 . 6 . then the sections were washed in tris - hcl . to eliminate the possible background due to non specific bonds , the sections were incubated for one hour at room temperature and under agitation in non - fat dry milk ( nfdm ) ( biorad , hercules calif .) at 6 ; in tris - hcl . overnight incubation followed at 4 ° c . with primary polyclonal antibody prepared in rabbit against htra1 human protein ( abeam , cambridge , uk ), diluted 1 : 100 in tris - hcl + nfdm 3 %. after some washings in tris - hcl , the sections were incubated for one hour with biotinylated anti - rabbit secondary antibody diluted 1 : 200 in tris - hcl - nfdm 3 %. after some additional washings in tris - hcl , the immunohistochemical reaction was detected with the abc method ( vector laboratories , burlingame , calif . ), ( 1 h at room temperature ) using dab ( 3 ′ 3 ′ diaminobenzidine tetrahydrochloride ) ( sigma - aldrich , milano , italia ) as chromogen . after being washed with tap water and then with deionized water , the sections were counterstained with mayer &# 39 ; s haematoxylin ( merck chemicals spa , milano , italia ) for approximately 2 minutes , placed under running water and dehydrated . then slides were mounted using the eukitt solution ( kindler gmbh and co ., freiburg , germany ). the normal bladder samples and the samples of subjects affected by cystitis showed immunostaining for htra1 prevalently homogeneous at urothelial level , present in all layers of the urothelium , but with higher positivity in the apical area of the cupoliform cells ( fig1 a , b ). the tunica media of the vesical vessels , as well as the muscular tunica of the bladder , were positive for htra1 ( fig1 a ). in the urothelium of papilliferous neoplasias with low malignity potential , htra1 proved to be weakly present in the cells of the basal urothelial layer ( fig1 c ), whereas the higher urothelial layers proved to be negative for htra1 . instead , positivity was still observed in the tunica media of stromal vessels , as well as in the muscular tunica of the bladder . the other neoplasias that were examined , meaning papilliferous neoplasias with growing malignity potential and infiltrating neoplasias , showed prevalently negative immunostaining for htra1 at urothelial cell level , whereas the muscular tunica of the bladder and the tunica media of the vessels showed weak positivity for htra1 ( fig1 d , e , f ). step 3 — preparation of protein extracts of normal and invasive tumoral bladder tissue samples for western blotting analysis portions of normal and tumoral bladder tissue , adjacent to the ones analyzed in immunohistochemistry , were immediately frozen in liquid nitrogen after the surgical sampling and then stored at − 80 ° c . the tumoral tissue samples only came from bladders of patients who underwent radical cystectomy because with turb the recovered tissue material was insufficient for the western blotting analysis . for the same reason , also the tissues taken with bladder biopsy from subjects affected by cystitis were not examined with such technique ( table 2 ). before being analyzed , the bladder tissue samples were defrosted and washed with 0 . 1 m ph 7 . 4 sodium phosphate buffer . at every washing , in order to avoid losing material , the samples were centrifuged at 800 g for 1 minute at 4 ° c . then , they were weighed and transformed in powder by means of manual breaking with pestle and mortar . every sample was resuspended in lysis buffer ( tris - hcl 20 mm , ph 8 . 0 , nonidet - p40 1 %, glycerol 10 %, nacl 137 mm , cacl2 1 mm , mgcl2 1 mm ) containing protease inhibitors ( sigma - aldrich ), setting the 0 . 1 g tissue / 1 ml buffer ratio . then , the samples were homogenized with potter ( ultra - turrax t8 , ika ®- werke , lille , france ) and centrifuged at 20000 g . the supernatants that were obtained , defined as raw extracts , were aliquoted and stored at − 80 ° c . protein concentration was measured with the bradford method ( 32 ). step 4 — analysis in western blotting of samples of normal bladder tissue and invasive tumoral bladder tissue . the protein extracts to be analyzed to quantitize the band relevant to the autopreolytic product of htra1 were prepared by denaturing 100 μg of total proteins with sample buffer 1 × ( tris - hcl 1 m ph 6 . 8 , sds 2 % e β - mercaptoethanol 1 %), keeping them boiling for 8 minutes . then , the samples were subjected to denaturing electrophoretic run in sds - page ( sodium dodecyl sulfate - polyacrylamide gel electrophoresis ) with laemmli method ( 33 ), making a constant current of 14 ma pass for 20 hours in a 10 % acrylamide gel . after terminating the electrophoretic run , proteins were transferred from the gel to a polyvynilidene fluoride ( pvdf ) membrane , using the tris - base 20 nm , glycine 150 mm , methanol 20 % buffer . the transfer was continued for one night , at constant voltage ( 30v ) at 4 ° c . then the membrane was washed with water , then in methanol for 15 - 20 seconds to fix the bands , then in water again , and finally in tbs - t ( tris - hcl 0 . 02m , ph 7 . 5 , nacl0 , 15 m , tween - 20 0 . 1 %). successively , it was immersed for 2 hours in agitation in the blocking buffer ( tbs - t + bsa 5 %) in order to increase the specificity of the antibody to its target protein . then the membrane was incubated for the night at 4 ° c . with monoclonal primary antibody directed against the htra1 / prss11 human protein ( amino acids 23 - 480 ) ( r & amp ; d systems , minneapolis , usa ) diluted 1 : 1000 in tbs - t + bsa 5 %. after 8 washings of 8 minutes each with tbs - t , the membrane was incubated for 2 hours at room temperature with anti igg murine secondary antibody conjugated to horseradish peroxidase ( hrp ) ( amersham sri , milano italia ), diluted 1 : 5000 in tbs - t + bsa 5 %, and then washed again for 8 times in one hour with tbs - t . to display the β - actin band , a protein used as housekeeping for data normalization , the primary monoclonal anti - actin antibody ( sigma ) was used and incubated for the night at 4 ° c . for the development of the chemoluminescence reaction , the ecl - plus solution ( amersham sri ) was used , acting on the membrane for 5 minutes . the membrane was dried , and then coated with film , inserted in an autoradiographic case together with the plate and exposed overnight . the development and fixation were carried out . fig2 a shows the western blotting image representative of the protein expression of the autoproteolytic form of htra1 in bladder tissue samples , which shows a significant constant difference in expression between normal and pathological samples . in particular , the autoproteolytic form of htra1 was significantly reduced in pathological tissue samples , where it appears extremely weak and difficult to detect in some cases . the protein expression in western blotting of β - actin in the same lysates of normal and pathological bladder tissues already analyzed to evaluate the levels of the autoproteolytic form of htra1 confirmed the gel homogenous loading ( fig2 b ). step 5 — calculation of molecular weight , densitometric analysis and statistical evaluation of signals , obtained in western blotting , of the autoproteolytic form of htra1 in normal and invasive tumoral bladder tissue samples . the equation and correlation coefficient r 2 were obtained from the calibration line constructed by considering the molecular weights ( p . m .) of each standard protein and the corresponding rf ( relative mobility ). inserting the rf measure of the band relevant to the autoproteolytic form of htra1 in the equation of the calibration line , the molecular weight was calculated , being 38 kda , as mentioned in literature ( 28 ). the images of the autoradiographic plates were acquired with a scanner and transformed in tiff format , eliminating any information on color , and then analyzed in optical densitometry with quantity one 4 . 6 . 1 ( biorad ) program . the quantity of protein relative to the band of htra1 at 38 kda was expressed in intensity values ( in grey scale ). β - actin was used as loading control and its expression values , obtained from the densitometric analysis , were used to normalize the densitometric data relevant to the expression of the autoproteolityc form of htra1 . the results were presented as mean ± standard deviation ( sd ). the statistical analysis of data was performed using the two - tailed t - test . from the semi - quantitative analysis of htra1 , we observed an expression reduction of the 38 kda for in pathological tissues compared to normal tissues and such a reduction proved to be highly significant ( p & lt ; 0 . 001 ) (***) ( fig3 ). step 6 — preparation of urine samples of healthy subjects , subjects with cystitis and patients affected by urothelial carcinoma for western blotting analysis . urine samples of healthy subjects , of subjects with cystitis and of patients affected by urothelial carcinoma ( tables 1 , 2 ) were collected in sterile containers at the first morning urination and , in case of carcinoma , taken the same day of the surgical operation ( turb or radical cystectomy ). immediately centrifuged for 15 minutes at 1500 g at 4 ° c ., they originated a pellet and a supernatant , the latter being stored at − 20 ° c . upon the analysis , urine was defrosted , centrifuged for 20 minutes at 3000 g at 4 ° c . and taken to the analysis laboratory of azienda ospedaliera universitaria ospedali ritniti “ umberto i - g . m . lancisi - g , salesi ( ancona ) to measure creatinine ( mg / dl ) and protein concentration ( g / l ). the urine samples were then concentrated with suitable devices ( amicon ultra - 4 and amicon ultra - 15 ) ( millipore , milan , italy ), choosing as reference a normal urine sample that , being previously concentrated and analyzed in western blotting , showed a quantizable signal . the all urine samples were concentrated for a variable number of times according to the relevant creatinine values related with the value of the reference sample ( normalization ). then concentrates were stored at − 20 ° c . step 7 — analysis in western blotting of urine samples of healthy subjects , subjects with cystitis and patients affected by urothelial carcinoma . the immunoprecipitation method was used for quantization of the native form of htra1 in urine . we used a 50 % suspension of sepharose spheres bound to g protein ( ge healthcare , bio - sciences ab , uppsala , sweden ) equilibrated in lysis buffer . for each urine sample to be immunoprecipitated , 50 μl of the sphere suspension were incubated for the night , at 4 ° c . and in rotation , with 3 pg of the monoclonal primary antibody directed against htra1 / prss1 1 human protein ( amino acids 23 - 480 ) ( r & amp ; d systems ). the sepharose spheres were first washed for 8 times with the lysis buffer , then , once collected , they were delicately resuspended in 50 μl of the same buffer and finally incubated for the night at 4 ° c . and in rotation with 225 μl of each concentrated urine . then , they were collected , washed for 3 times with the lysis buffer and centrifuged at 13000 g for 1 minute for pelleting . the bound proteins were eluted from the spheres through incubation with 45 μl of glycine 100 mm , ph 2 . 5 for 30 minutes at room temperature and in agitation . elutes ( 40 μl ) were then collected , denatured in sample buffer 1 ×, neutralized with soda and boiled for 8 minutes . urine immunoprecipitates were loaded in a 10 % acrylamide gel and transferred on pvdf membrane . then , antibody reaction was carried out with monoclonal primary antibody directed against the htra1 / prss1 1 human protein ( amino acids 23 - 480 ) ( r & amp ; d systems ), and detection of bands was performed by means of chemoluminescence , following the same protocol used for tissues . the expression of urinary htra1 in its native form ( 53 kda ) was positive , although in a variable way , in 100 % of the patients with urothelial carcinoma who underwent radical cystectomy or turb ( fig4 ), whereas was practically absent in all normal cases and in subjects with cystitis ( fig5 ), thus suggesting that htra1 is not generally involved in inflammatory pathologies . step 8 — densitometric analysis and statistical evaluation of signals , obtained in western blotting , of the native form of htra1 in urine samples of healthy subjects , subjects with cystitis and patients affected by urothelial carcinoma . by means of densitometry , the intensity values ( in grey scale ) of the native form of htra1 were calculated in all urine samples that were analyzed . the values were submitted to the one - 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