Patent Application: US-201515529937-A

Abstract:
the present invention concerns the use of protein biomarkers for use in facilitating in the prognosis and / or treatment regime of bladder cancer . in particular , the invention relates to the use of shed protein fragments , such as fragments of epithelial cell adhesion molecule and / or epidermal growth factor receptor detected in a sample of urine , as biomarkers for use in facilitating the prognosis and / or treatment regime of urothelial bladder cancer .

Description:
the present invention will now be further described by way of example and with reference to the attached tables and figures which are not to be construed as limiting . fig1 shows the urinary egfr in ubc patients . the left hand panel shows urinary egfr data for patients stratified according to stage and the right hand panel according to grade . fig2 shows the effects of ultracentrifugation and pma stimulation on cell line secretomes . cells were incubated overnight in serum free media ± 200 nm pma and egfr measured in the conditioned media by elisa pre - ultracentrifugation ( black bars = control , dark grey bars = pma ) or post - ultracentrifugation ( light grey bars = controls , white bars = pma ). data are presented relative to the control for each cell line . the insert shows an egfr western blot of hb - cls - 2 cell lysate and secretome ( w = whole , u = ultracentrifuged ). fig3 shows the prognostic value of urinary egfr . kaplan - meier curves are shown for ubc - specific survival with patients stratified according to low / high urinary egfr (& gt ; 630 pg / mg creatinine ). fig4 shows the relationship between urinary egfr and epcam in ubc : the creatinine - normalised concentrations of egfr and epcam in the urine of 113 patients with mibc . fig5 shows the prognostic value of urinary egfr and epcam . kaplan - meier curves are shown for bladder cancer - specific survival with patients stratified according to low / high urinary egfr and epcam . since the original egfr study detailed herein and with reference to fig1 - 5 , the patient cohort has been expanded to include 911 bladder cancer patients . kaplan - meier survival curves for bladder cancer specific survival in the expanded cohort stratified into quartiles dependent on egfr , epcam and hai - 1 concentration are shown in fig6 - 8 . the cell lines used in this study were selected on the basis that they exhibit diverse mutation profiles and may capture some of the heterogeneity of ubc ( supplementary data , table 1 ). urine samples were prospectively collected for biomarker research between 2006 and 2009 as part of the bladder cancer prognosis programme ( bcpp , ethics approval 06 / mre04 / 65 ) ( zeegers et al , 2010 ). patients were enrolled on the basis of initial cystoscopic findings suggestive of primary ubc . all ubc patients were newly - diagnosed , had not received treatment for ubc prior to urine collection , and were subsequently treated according to current standard practice . inclusion and exclusion criteria are detailed elsewhere ( zeegers et al , 2010 ). samples were placed on ice , centrifuged at 2000 rpm for 10 minutes within 8 hours of collection , and supernatant stored at − 80 ° c . since patient recruitment occurred prior to histopathological confirmation of ubc , a proportion of patients were ultimately diagnosed with non - malignant conditions and these serve as non - cancer ‘ controls ’. all patients were followed - up to july 2014 with causes of death notified to the bcpp study office . secretomes were harvested and analysed by shotgun proteomics as described previously ( shimwell et al , 2013 ), except that secretomes were collected for each cell line with or without 200 nm phorbol 12 - myristate 13 - acetate ( pma ). proteins were digested with trypsin and stable isotope labelled ( dimethylation with formaldehyde / deuterated formaldehyde ) to allow relative protein concentrations ± pma to be calculated . cells were grown to confluence ( apart from rt4 cells which grow as islands ). adherent cells were then washed 4 times with serum - free media and incubated overnight in serum - free media ± 200 nm phorbol 12 - myristate 13 - acetate ( pma ). the conditioned media were centrifuged at 1500 × g for 20 minutes prior to filter - aided tryptic digestion . proteins were reduced with 20 mm dtt in 8m urea , 1 % chaps , 100 mm teab ( 1 hour at room temperature ), alkylated with 40 mm iodoacetamide and then concentrated in 30 kda centrifugal filters . following 4 washes with 100 mm triethylammonium bicarbonate , sequencing grade trypsin was added to the filters ( 1 : 50 trypsin : protein ) and incubated at 37 ° c . overnight . the peptides were centrifuged through the filters and their primary amines dimethylated by incubating with 25 mm sodium cyanoborohydride and 0 . 2 % formaldehyde ( control secretome ) or 0 . 2 % deuterated formaldehyde (+ pma secretome ) for 30 min at room temperature . following quenching with 100mm ammonium bicarbonate the samples were fractionated by mixed - mode hplc and analysed by lc - ms / ms and the data searched against swissprot human and bovine sequence databases using mascot . multiple datasets were combined using proteinscape 3 ( bruker daltronics ) and filtered at a 1 % protein false discovery rate . light and heavy dimethylation of n - termini and lysine side - chains were included as variable modifications and relative quantitation was performed using warp - lc software ( bruker daltonics ). egfr was measured with a sandwich elisa utilising goat polyclonal antibodies against the extracellular domain of egfr ( r & amp ; d systems , cat no . dyc1854 - 5 ). between 2 and 100 μl of secretome or 50 μl of urine plus 50 μl of 1 % bsa in pbst was used per well . urinary egfr is expressed as pg egfr per mg creatinine . data was downloaded from http :// www . proteinatlas . org and a list of 54 proteins overexpressed in ubc drawn up on the basis that they exhibited high or medium staining in at least 4 cases ( out of 11 or 12 cases depending on the protein ) and low or undetectable staining in normal urothelium . urinary egfr levels are presented as medians and statistical significance was calculated using mann - whitney tests . ubc - specific survival was defined as the time from registration into the bcpp study to date of death from ubc . patients were censored at the date last known to be alive or date of ubc - related death . univariable cox proportional hazards models were employed ( alpha 0 . 1 ) to identify factors to be included in a cox multivariable model ; significance was set at 0 . 05 . analysis was done in stata 12 . 1 ( statacorp , college station , tex ., usa ). in total , 2104 proteins were identified in the conditioned media of one or more cell lines ( table 2 ). to select candidate biomarkers we considered proteins released by at least 3 of the 8 cancer cell lines and also reported to be overexpressed in ubc in the human protein atlas . this generated a shortlist of 5 candidates : egfr , g6pd , prdx6 , lypd3 and fbln1 . egfr was unique in that it was the only one of these 5 proteins that was identified in the cancer cell line secretomes and not in the urotsa secretome . tryptic peptides from egfr were detected in 5 of the 8 ubc cell lines with a tendency for more peptides ( suggestive of a higher egfr protein concentration ) in the cell lines derived from higher grade tumours ( table 2 ). the levels of egfr in the secretomes were subsequently measured by elisa and found to reflect the number of peptides identified . egfr secretome levels also reflected levels in the corresponding cell lysates as determined by western blotting ( data not shown ). egfr was measured in the urine of 436 ubc patients and 60 non - cancer controls . the data are summarised in table 3 . the median urinary egfr concentration was 153 pg / mg creatinine in the non - cancer controls and was not significantly increased in patients with pta ubc . there were significantly higher levels of egfr in the urine of patients with pt1 or pt2 + ubc ( 224 and 317pg / mg creatinine , respectively ). the data for individual patients stratified according to stage and grade is shown in fig1 . high levels of urinary egfr were observed in a proportion , but not all , patients with high grade and high stage ubc . defining the upper limit of the normal reference range as the mean + 2sd gave a threshold of 630 pg egfr / mg creatinine , and the percentage of positive cases is 7 , 4 , 13 , and 27 in non - cancer controls , pta , pt1 and pt2 + ubc , respectively . peptides from the extracellular domain , but not the intracellular domain , of egfr were identified in secretomes from mgh - u3 , rt112 and 5637 cell lines . vm - cub - 1 and hb - cls - 2 secretomes contained the highest levels of egfr , and peptides from both the intracellular and extracellular domains were identified , however , both spectral counting and peptide intensity suggested a higher concentration of the extracellular domain than the intracellular domain i . e . the extracellular domain of egfr is shed into the conditioned media and membrane - bound egfr is also present . to test this hypothesis we ultracentrifuged the secretomes at 136 , 000 × g for 90 minutes to pellet membranes and membrane - bound proteins and measured the egfr remaining in the supernatants . on average , across the secretomes , 73 % of the egfr remained in the supernatant confirming that most of the egfr is a soluble rather than membrane - bound form ( fig2 ). western blotting of the proteins released by hb - cls - 2 cells indicated that the predominant form of egfr in the secretome has a molecular weight of approximately 100 kda . a small amount of full - length egfr ( approximately 170 kda ) is also present but is removed by ultracentrifugation ( fig2 ). the ratio of heavy to light egfr peptide intensities in the mass spectra suggested that egfr is released constitutively but can be further stimulated by pma , and this was confirmed by elisa : an increase in the amount of egfr released ( both total and soluble forms ) was seen with most of the cell lines and was as high as 4 - 5 - fold in some cases ( fig2 ). ultracentrifugation was also used to test whether urinary egfr was shed extracellular domain or membrane - bound egfr : the egfr concentrations of 12 urine samples were measured pre - and post - ultracentrifugation . on average , 93 . 5 % ( sd 7 . 8 %) remained in the supernatant , indicating that the predominant form of egfr in urine is soluble extracellular domain . univariable analyses included age ( years ), multiple tumours ( 1 vs 2 +), grade ( 1 , 2 vs 3 ), size of largest tumour ( p & lt ; 3 cm vs & gt ; 3 cm ), cis ( present vs absent ), stage ( pta or pt1 vs pt2 +), sex ( male vs female ) and urinary egfr ( normal vs elevated ). grade , stage , age , tumour size and cis all reached statistically significance and were included in the multivariable analysis . in univariable analysis egfr was found to be highly significant ( hr 6 . 9 90 % ci 4 . 7 , 10 . 1 , p & lt ; 0 . 001 ) and this can be seen in kaplan - meier curves for ubc - specific survival in both nmibc and mibc cases ( fig3 ). multivariable analysis showed that elevated urinary egfr ( 624 pg egfr per mg creatinine ) is prognostic ( hr 2 . 9 95 % ci 1 . 1 , 4 . 6 : p & lt ; 0 . 001 ) in addition to grade and stage for ubc - specific survival ( table 4 ). we have previously reported that the urinary concentration of the extracellular domain of epcam is associated with a shorter ubc - specific survival time ( bryan et al , 2014b the entire contents of which are herein incorporated by reference , and which is appended hereto by way of reference ). there is little correlation between the secretome levels of epcam and egfr across ubc cell lines ( table 2 ), suggesting that using these proteins might act as prognostic markers independently of one another . similarly there is little correlation between the urinary concentrations of egfr and epcam in ubc patients : many patients have high levels of either epcam or egfr , with 33 mibc patients positive for egfr , 45 positive for epcam and 59 positive for one or both proteins ( 52 % of the mibc patients ) ( fig4 ). multivariable modeling including both biomarkers was conducted . grade and stage both remained highly prognostic ( p = 0 . 011 , p & lt ; 0 . 001 respectively ), as did both egfr ( hr = 6 . 69 95 % cl 3 . 41 , 13 . 12 p & lt ; 0 . 001 ) and epcam ( hr = 2 . 13 95 % cl 1 . 19 , 3 . 82 p = 0 . 011 ) ( table 5 ). the risk associated with having elevated levels of both biomarkers was found to be greater than those with only epcam elevation , but less than those with only elevated egfr ( p = 0 . 001 ) ( fig5 ). combining proteomic analysis of ubc cell line secretomes and immunohistochemistry data available in the human protein atlas identified egfr as a potential urinary biomarker for ubc . we then tested the diagnostic and prognostic utility of urinary egfr in a cohort of 496 patients with long - term clinical follow - up . urinary egfr is elevated in 27 % of mibc cases and 17 % of grade 3 ubcs but normal levels are observed in pta and low - grade disease ; thus , overall , urinary egfr is not a good diagnostic marker for ubc . however , urinary egfr is a strong independent prognostic indicator of ubc - specific survival . urinary egfr could have utility for rapidly identifying patients with the most aggressive disease , and expediting their subsequent investigation and management . shedding of egfr from cancer cell lines ( although not ubc cell lines ) has been reported to be related to overexpression , to be stimulated by pma ( and hence may be under the control of protein kinase c ), and to be blocked by metalloprotease inhibitors ( perez - torres et al , 2008 ). adamczyk reported that pancreatic cancer cell lines release both exosomal ( full length , 170 kda ) and ectodomain ( 110 kda ) forms of egfr ( adamczyk et al , 2011 ). the evidence suggests that egfr ectodomain is shed as the result of proteolytic cleavage and that it is probably cleavage of an alternatively spliced 3 kb transcript of egfr that generates soluble ectodomain ( wilken et al , 2013 ). although this splice variant lacks both the transmembrane and intracellular domains it seems that it remains membrane associated until cleaved ( wilken et al , 2013 ). our ubc cell line data is consistent with the body of evidence suggesting that both full - length and ectodomain egfr are released from cancer cells by a regulated mechanism involving proteolytic cleavage . soluble egfr has been detected in plasma and investigated in several cancers . however , plasma levels appear to be lower in cancer patients than healthy controls , are not related to egfr expression in tumours and there is little evidence of diagnostic , predictive or prognostic value ( baron et al , 2003 ; hudelist et al , 2006 ; lemos - gonzález et al , 2007 ; müller et al , 2006 ; zampino et al , 2008 ). although egf has been measured in the urine of ubc patients ( chow et al , 1997 ), the only case of egfr being measured in urine that we have encountered looks at a number of distal tumour sites and not ubc ( witters et al , 1995 ). thus , we believe that the current work is the first exploration of soluble urinary egfr as a biomarker for ubc , with a much clearer relationship between urinary egfr and ubc than in studies of plasma egfr in other solid malignancies . egfr has been reported to be overexpressed in many epithelial cancers and was reported as a poor prognostic indicator in ubc as early as 1990 ( neal et al , 1190 ), representing a late event in the progression of ubc ( lipponen & amp ; eskelinen , 1994 ). more recently , egfr overexpression has been shown to be a characteristic of basal - like aggressive mibc , and in mouse models these tumours respond to anti - egfr therapy ( rebouissou et al , 2014 ). several clinical trials using anti - egfr therapies in combination with chemotherapy or radiotherapy in mibc are currently underway . without wishing to be bound by theory , the urinary levels of egfr may be due to the level of expression in tumour cells , and / or due to shedding of the egfr ectodomain . thus , urinary egfr could be a facile indicator of a patient &# 39 ; s suitability to be treated with anti - egfr therapy . we present the first description of the prognostic value of soluble urinary egfr in ubc , both alone and in combination with soluble urinary epcam ( bryan et al , 2014b the entire contents of which are herein incorporated by reference ). these elisa - based tests are easy and reproducible , and thus have significant potential clinical utility and we intend to evaluate this potential prospectively in ubc . mechanisms of ectodomain shedding may add an extra layer of complexity to the molecular pathology of ubc that may not be uncovered by genomic approaches and appear to unmask potentially important prognostic markers . better understanding of these phenomena may also reveal new therapeutic targets , targets that are desperately needed for ubc ( bryan et al , 2014a ). chow n , liu h , lee e , chang c , chan s , cheng h , tzai t , lin j ( 1997 ) significance of urinary epidermal growth factor and its receptor expression in human bladder cancer . anticancer res 17 : 1293 - 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