Patent Application: US-57075104-A

Abstract:
the present invention relates to methods of detecting , monitoring the efficacy of treatment of , and assessing the severity of ovarian cancer , by assessing the concentration of haptoglobin - 1 precursor in a sample of biological fluid . the invention also relates to a kit comprising an antibody or nucleic acid probe specific for haptoglobin - 1 precursor for use in the diagnosis of ovarian cancer , monitoring the efficacy of treatment of ovarian cancer , or assessing the severity of ovarian cancer .

Description:
it is to be clearly understood that this invention is not limited to the particular materials and methods described herein , as these may vary . it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only , and it is not intended to limit the scope of the present invention , which will be limited only by the appended claims . as used herein , the singular forms “ a ”, “ an ”, and “ the ” include the corresponding plural reference unless the context clearly dictates otherwise . thus , for example , a reference to “ a protein ” includes a plurality of such proteins , and a reference to “ a molecule ” is a reference to one or more molecules . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention , the preferred materials and methods are now described . in the claims which follow and in the preceding description of the invention , except where the context requires otherwise due to express language or necessary implication , the word “ comprise ” or variations such as “ comprises ” or “ comprising ” is used in an inclusive sense , i . e . to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention . when a range of values is expressed , it will be clearly understood that this range encompasses the upper and lower limits of the range , and all values in between those limits . “ haptoglobin - 1 ” refers to the mature glycolysated tetramer of molecular weight approximately 90 kd . “ haptoglobin - 1 precursor ” refers to the single chain precursor protein of molecular weight approximately 38 kd , which includes the 18 amino acid signal sequence . “ immunoreactive haptoglobin - 1 precursor ” refers to haptoglobin - 1 precursor detected using monoclonal antibody directed to mature haptoglobin - 1 . 1 - de one - dimensional electrophoresis 2 - de two - dimensional electrophoresis ir immunoreactive maldi - tof matrix - assisted laser desorption interferometry - time of flight ms mass spectroscopy ms / ms tandem mass spectroscopy seldi - tof ms surface - enhanced laser desorption ionization time - of - flight mass spectrometry tof ms time of flight mass spectrometry we have found that haptoglobin precursor is present in ascites and in the serum of grade 1 , grade 2 and grade 3 ovarian cancer patients . in ovarian cancer tissues , immunoreactive haptoglobin - 1 precursor is present in the epithelial cells , stroma and ovarian vessels . haptoglobin - 1 precursor is & gt ; 90 % homologous to mature haptoglobin . hence a monoclonal antibody against haptoglobin is able to detect immunoreactive haptoglobin - 1 precursor . these data are consistent with the hypothesis that overexpression of haptoglobin - 1 precursor is an early event in the onset of ovarian cancer . thus enhanced expression of haptoglobin - 1 precursor may represent a condition of acute response , which is a pre - requisite for tumour progression . without wishing to be limited to any proposed mechanism , we believe that since most secretory proteins are initially synthesized as larger precursors with an extended nh 2 - terminal sequence which is cleaved at the late stage of the secretory process , either within the golgi complex or in related vesicles , the elevated levels of the haptoglobin - 1 precursor in the serum of cancer patients may result from defective intracellular processing which is specific to cancer cells . transcriptional profiling , or the related serial analysis of gene expression , subtractive hybridization and differential display technologies , has identified fourteen candidate ovarian cancer markers ( mok et al , 2001 ; schummer et al , 1999 ). among these proteins , mesothelin and kallikrein 10 were identified by the monoclonal antibody and candidate gene approaches , respectively ( scholler et al , 1999 ; luo et al , 2001 ). mesothelin is elevated in the serum of 76 % of ovarian cancer patients ( diamandis et al , 2000 ), and kallikrein 10 is elevated in 56 % of ovarian cancer patients ( luo et al , 2001 ). it has been suggested that tests for mesothelin and / or kallikrein 10 may be able to complement the ca125 test , increasing the prospect of detecting ovarian cancer at an early , curable stage . gene array technology has been used to identify prostasin , a serine protease , previously identified in prostatic secretions , osteopontin , a secreted bone morphogen , and creatine kinase b , a marker for renal and lung cancers , as being elevated in serum from patients with ovarian cancer ( mok et al , 2001 ; kim et al , 2002 ). these data indicate that screening approaches at the dna or rna level may be able to identify a series of markers which also have the potential to complement the currently used ca125 test and to provide increased specificity and sensitivity . we have recently identified a cell - free immunoreactive form of integrin - linked kinase ( ilk ) as a marker for ovarian cancer which is highly correlated with the stage of the cancer . see international patent application no . pct / au03 / 01058 in the name of the royal women &# 39 ; s hospital , filed on 20 august 2003 . one or more additional markers for ovarian cancer , such as ilk ( pct / au03 / 01058 ), ca125 ( mackay and creasman , 1995 ), tadg - 12 ( underwood l j , 2000 ), or the more recently - described markers , serotransferrin ( kawakami et al , 1999 ), neutrophil gelatinase associated lipocalin ( kjeldsen et al , 1994 & gt ;, soluble cd163 ( baeton et al , 2003 ) and gc - globulin ( jorgensen et al , 2004 ) may be used in the methods of the invention as an adjunct to the detection of haptoglobin - 1 precursor . two - dimensional electrophoresis and mass spectrometry first dimension separation : twenty five μg of serum protein were mixed with rehydration buffer ( 7 m urea , 2 m thiourea , 100 mm dithiothreitol ( dtt ), 4 % ( 3 -[( 3 - cholamidopropyl ) dimethylammonio ]- 1 - propanesulfonate ) ( chaps ), 0 . 5 % carrier ampholytes , 0 . 01 % bromophenol blue ( bpb ) 40 mm tris and ph 4 - 7 ) to a final volume of 200 μl , and incubated for 1 h at room temperature . this mixture was then applied to a ready strip ( 11 cm , ph 4 - 7 ; bio - rad laboratories , usa ) and actively rehydrated at 50 v at 20 ° c . for 16 h . serum proteins were subjected to isoelectric focusing at 250 v for 15 min ; the voltage was then slowly ramped up to 8000 v for 150 min , and then maintained at 8000 v for a total of 35000 vh / gel ( i . e . a total of 42000 vh per gel ). the ready strips were then stored at − 80 ° c . prior to second dimension separation . second dimension separation : ready strips from the first dimension separation were equilibrated in 5 ml of equilibration buffer ( 50 mm tris - hcl ph 8 . 8 , 6 m urea , 30 % glycerol , 2 % sodium dodecyl sulfate ( sds ), 0 . 01 % bpb , 2 mm tributyl phosphine ( tbp )). strips were rinsed in tris - glycine - sds running buffer ( 25 mm tris , 192 mm glycine , 0 . 1 % w / v sds ; ph 8 . 3 ) and then applied to the top of a 10 % tris - hcl precast criterion gel ( bio - rad laboratories , usa ). low melting point agarose ( 0 . 5 % in running buffer containing bpb ) was layered on top of the strip . molecular weight markers ( 150 , 100 , 75 , 50 , 37 and 25 kda ) were run simultaneously . gels were electrophoresed at 10 ma / gel for 1 h , 20 ma / gel for 2 h and then 30 ma / gel for 30 min . gels were then fixed in methanol / acetic acid ( 40 %/ 10 % in dh 2 o ) for 1 h at room temperature and incubated in sypro ruby ® ( bio - rad laboratories , usa ) for 16 h at room temperature on a rocking platform . gels were de - stained for 1 h in methanol / acetic acid ( 10 %/ 7 % in dh20 ), imaged using a bio - rad fx imager at 100 nm resolution , and analyzed using pdquest version 6 software ( bio - rad laboratories , usa ). the computer program identified protein spots from the digitized images of the gel . analyses of some serum samples were repeated three times to assess the variability between the experiments on different gels . mass spectrometry : following the imaging , the gels were stained with coomassie blue to enable visual identification and isolation of the protein bonds . coomassie stained bands were excised from the gel and digested with trypsin . mass spectrometry experiments were performed on an ettan maldi - tof instrument ( amersham bioscience , uk ) and api qstar pulsar i mass spectrometer ( applied biosystems , mds sciex , framingham , usa ). tofms data were searched via pepsea server , which is included in the analyst software ( applied biosystems , mds sciex , framingham , usa ). tandem ms data were searched via the mascot search engine . the invention will now be described in detail by way of reference only to the following non - limiting examples and drawings . human serum samples were treated with a mixture of affigel - blue and protein a ( 5 : 1 ) in the form of a spin column ( bio - rad laboratories , usa ). the spin columns contained a mixture of affi - gel blue and protein a , which selectively binds and removes albumin and immunoglobulin . the spin columns were washed twice with 1 ml of binding buffer ( 20 mm phosphate buffer , ph 7 . 0 ) by centrifugation for 20 sec at 1000 × g . 50 μl of serum was added to 150 μl of binding buffer , mixed by vortexing , and loaded on the spin columns . following incubation at room temperature for 1 h , columns were centrifuged for 20 sec at 1000 × g to collect the eluate . the columns were washed with 200 μl of binding buffer and combined with the first eluate to form the depleted serum sample . the total protein concentration of the combined eluate was determined . the eluate was stored at − 80 ° c . until further analysis . fig1 a demonstrates a typical 2 - de human serum profile visualized by sypro ruby staining . more than 300 proteins were detected and localized between pi 4 - 7 and molecular mass range of 20 - 200 kda . the albumin smear at around 68 kda was present in untreated serum , but within 1 h of affi - gel blue and protein a treatment significant loss of albumin was achieved , with no significant loss of other proteins displayed . concomitant with the removal of albumin there was a significant enhancement in the staining intensity of several protein spots , as shown in fig1 b . these results indicate that affi - gel blue and protein a treatment of human serum results in the removal of high abundance albumin , thereby increasing the detection of low abundance proteins which would have remained obscured in the presence of albumin . we have implemented this approach of albumin clearance for the identification of differentially - expressed low abundance proteins in the serum of ovarian cancer patients . expression of haptoglobin - 1 precursor in serum and ascites from ovarian cancer patients proteomic analysis and mass spectrometry were used to evaluate the expression of haptoglobin - 1 precursor in the serum of normal healthy women and of ovarian cancer patients . cancer patients were graded according to standard histological methods ( silverberg , 2000 ). the mean age of women in the control group and women with ovarian cancer was 47 and 62 years respectively . whole blood ( 10 ml ) was collected by venepuncture into plain collection tubes for serum ( blood was allowed to clot at room temperature for 30 min ). samples were centrifuged at 2000 g for 10 min after which serum was collected . an aliquot ( 100 μl ) was removed for the determination of total protein . serum was stored at 80 ° c . until analyzed . serum samples from 8 normal female subjects and 19 ovarian cancer patients were analysed for the expression of haptoglobin - 1 precursor . of the patients , 6 were grade 1 , 8 were grade 2 , and 24 were grade 3 . ascitic fluid of ovarian cancer patients was also tested for haptoglobin - 1 precursor expression . haptoglobin - 1 precursor expression was detected in serum and ascitic fluid by proteomic analysis and by western blotting under non - reducing conditions , using a monoclonal antibody against mature haptoglobin ( sigma , st louis , usa ). haptoglobin - 1 precursor is & gt ; 90 % homologous to mature haptoglobin . hence the monoclonal antibody against haptoglobin is able to detect immunoreactive haptoglobin - 1 precursor . whole blood ( 2 ml ) was collected by venepuncture into plain collection tubes , and allowed to clot at room temperature for 30 min . samples were then centrifuged at 2000 g for 10 min , after which serum was collected . an aliquot ( 100 μl ) was removed for the determination of total protein . serum was stored at − 80 ° c . until analysed . blood specimens were thawed at room temperature , and two - dimensional electrophoresis was performed on the specimens . fig2 shows the results of proteomic analysis of expression of haptoglobin - 1 precursor in the serum of normal subjects and in grade 1 , grade 2 and grade 3 ovarian cancer patients . fig3 shows the results of proteomic analysis of expression of haptoglobin - 1 precursor in ascitic fluid from ovarian cancer patients . serum protein profile of ovarian cancer patients at different histological grades protein profiles of the serum of grade 1 ( n = 6 ), grade 2 ( n = 8 ) and grade 3 ( n = 24 ) ovarian cancer patients were analyzed by 2 - de and visualized by staining with sypro - ruby . protein profiles of replicate sets of samples from cancer patients were compared with the serum of normal healthy women ( n = 8 ) using pdquest software , and the gaussian profiles are shown in fig4 . the quantitative evaluation of the differentially expressed serum proteins in normal vs grade 1 , grade 2 or grade 3 ovarian cancer patients was performed using student &# 39 ; s t - test . significant differences in the overall profiles of serum proteins were obtained in grade 1 , 2 and 3 ovarian cancer patients compared to normal healthy volunteers . compared to normal serum , twenty - four proteins were differentially expressed in the serum of grade 1 ovarian cancer patients ( fig4 a ). of these proteins , fifteen proteins were up - regulated by two - fold , four proteins by five - fold and two proteins by ten - fold . in contrast , one protein was down - regulated by two , five and ten - fold respectively . in grade 2 cancer patients , differential expression of thirty - one proteins was observed of which twenty - five were up - regulated by two - fold , four by five - fold and two by ten - fold . analysis of serum from grade 3 cancer patients demonstrated two - fold down - regulation of thirteen proteins out of twenty - five differentially - expressed proteins ( fig4 b and 4 c ). six proteins were up - regulated by two - fold , three by five - fold and two by ten - fold respectively ( p & lt ; 0 . 05 ). some proteins were found to be uniquely expressed only in the serum of a specific pathological grade of cancer patients , and some proteins were not consistently expressed among the three histological grades of cancer patients . to ensure consistency in the observed differential expression profile , analyses of serum samples from the same patient prepared on three different days were repeated three times , and investigated to eliminate confounding factors that may arise from sample handling . no substantial variation in the profile of protein spots of the same sample repeated on different days was detected . among the differentially - expressed serum proteins , ten proteins were found to be consistently expressed in grade 1 , 2 and 3 cancer patients ( p & gt ; 0 . 05 ). six of these proteins , which had approximate molecular weights of 40 kda and pi of 5 . 9 - 6 . 6 , were significantly over - expressed in the serum of grade 1 , 2 and 3 ovarian cancer patients . these were selected for further analysis and identification . the six proteins found in example 3 to be over - expressed in ovarian cancer patients were identified by nano - electrospray quadrupole quadrupole time of flight mass spectrometry ( esiq ( q ) tof ms ) and matrix - assisted laser desorption ionization time of flight mass spectrometry ( maldi - tof ms ) analysis . mass fingerprinting spectra from the six proteins , shown in fig5 , showed identical fragmentation patterns of the peptides , suggesting the possibility of a series of post - translational modifications of a single protein separating at different pi and / or molecular mass values . ms / ms analysis of the six proteins confirmed their identity as isoforms of haptoglobin - 1 precursor ( swissprot accession number p00737 ), a protein with a molecular mass of 38 . 42 kda and pi of 6 . 1 - 6 . 6 which shares 90 % homology to the liver glycoprotein haptoglobin , which is present in normal serum ( beutler et al , 2002 ). the amino acid sequences of these peptides are summarized in table 1 . the peptide sequence obtained encompassed amino acid sequences corresponding to different regions of haptoglobin - 1 precursor . differences in the glycosylation pattern of the protein have the potential to change both the pi and molecular mass of the protein , and different sialylated forms of haptoglobin have been demonstrated in normal serum using the 2 - de approach ( wilson et al , 2002 ). our results , which demonstrate that six different isoforms of haptoglobin - l precursor are present in the serum of ovarian cancer patients , are consistent with these observations . further confirmation that these proteins were isoforms of haptoglobin - 1 precursor was obtained by western blotting using monoclonal anti - human haptoglobin antibody . the six isoforms of haptoglobin - 1 precursor were detected by 1 - de or 2 - de and western blotting as a chain of protein spots with slightly different molecular masses and different pis . the native protein has 90 % homology to the precursor , so monoclonal - anti - haptoglobin antibody is expected to visualize haptoglobin isoform precursors on 2 - de western blot . for western blots , serum samples were incubated with 5 volumes of laemmli buffer . specimens containing equal amounts of protein ( 60 μg ) were electrophoresed on a 10 % sds - page gels under non - reducing conditions , and then transferred to nitrocellulose membranes . membranes were probed with primary haptoglobin antibody ( sigma , usa ) followed by peroxidase - labelled secondary antibody ( amersham , uk ) and visualised by the ecl detection system ( amersham , uk ) according to the manufacturer &# 39 ; s instructions . the results are shown in fig6 and 7 . fig6 a shows the 1 - de western profile of haptoglobin molecules in the serum of healthy volunteers and ovarian cancer patients . the antibody predominantly recognizes three bands at approximate molecular weights 20 , 40 and 70 kda . interestingly , the expression of the proteins identified by anti - haptoglobin antibody at 40 and 70 kda was greater in grade 1 and 3 cancer patients than in healthy adults . consistent with this , high reactivity was observed with the set of six proteins at 40 kda molecular weight by 2 - de western blot ( fig6 b , 6 c and 6 d ). similar results were obtained using 2 - de and western blotting , as illustrated in fig7 . reactivity was also observed with two additional proteins at molecular weight 20 and 70 kda , and was consistent with the 1 - de profile . the identity of the proteins at molecular weights 20 and 70 kda is not known , and is under investigation . the six isoforms of haptoglobin - 1 precursor , exhibited as a chain of protein spots with slightly different molecular mass and different pis , suggest the presence of post - translational modifications . these results indicate that quantification of haptoglobin - 1 precursor expression in the serum of ovarian cancer patients , for example by elisa or radio - immunoassay , can be used as a diagnostic marker for screening purposes . paraffin - processed archival tissues were obtained from the department of pathology , royal women &# 39 ; s hospital , melbourne . these included normal ovaries ( n = 6 ) needed for control comparisons , which were removed from patients undergoing surgery as a result of suspicious ultrasound images , palpable abdominal masses and family history . the pathology diagnosis and tumour grade was determined by two staff pathologists in the department of pathology , royal women &# 39 ; s hospital , melbourne . the classification of the tumours was carried out as part of the clinical diagnosis . histological grading of ovarian carcinoma was performed by the method described by silverberg ( 2000 ). tissue sections were cut at 4 μm thickness , mounted on poly - l - lysine coated slides and incubated for 1 h at 60 ° c . sections were brought to water through 3 changes each of xylene and ethanol . antigen unmasking was performed using citrate buffer ( ph 6 . 0 ) in a microwave oven . endogenous peroxidases were removed using 3 % hydrogen peroxide in methanol , and endogenous biotin activity was blocked using a sequence of diluted egg white ( 5 % in distilled water ) and diluted skim milk powder ( 5 % in distilled water ). sections were incubated for lh in anti - haptoglobin monoclonal antibody ( sigma , st louis usa ) diluted 1 / 10 000 in 1 % bsa in tris buffer ( 100 mm , ph 7 . 6 ). antibody binding was amplified using biotin and streptavidin hrp ( dako , denmark ) for 15 min each , and the complex visualized using diaminobenzidine . nuclei were lightly stained with mayer &# 39 ; s haematoxylin . an isotype igg1 , suitably diluted , was substituted for the antibody as a negative control . sections were assessed microscopically for positive diaminobenzidine staining . the intensity of haptoglobin expression was scored in a blind fashion as negative , weak , moderate or strong immunoreactivity . in addition to the type of staining , the tissue and cellular distribution of staining was determined . fig8 shows the results of an immunohistochemical comparison between samples of normal ovarian epithelium and of serous and endometrioid ovarian tumours at different grades . no immunoreactive haptoglobin - 1 precursor was detected in normal ovarian surface epithelium or stroma , as shown in fig8 a . in grade 1 and grade 3 serous and endometrioid ovarian tumours , shown in fig8 b , 8 c and 8 d respectively , high expression of immunoreactive haptoglobin - 1 precursor was detected in epithelium , stroma and ovarian vessels . the staining was mostly cytoplasmic , with the majority of the staining being observed in scattered cell groups . tumours with a glandular pattern tended to have more staining . strong staining was evident in areas with myxomatous stroma or vascular spaces , as well as ovarian vessels . without wishing to be limited by any proposed mechanism , we believe that these results suggest that haptoglobin precursor is expressed by ovarian tumour cells , but that elevated haptoglobin precursor concentrations in the serum of ovarian cancer patients are most probably of hepatic origin . the efficacy of detecting the expression of haptoglobin - 1 precursor in samples from biological fluid from ovarian cancer patients before and after six cycles of chemotherapy was assessed . the chemotherapy treatment comprised a conventional combination regimen consisting of carboplatin ( auc 5 )/ taxol ( 175 mg / m 2 body weight ) following surgery . the combination drugs were given to patients every three weeks by intravenous infusion . the pre - chemotherapy sample was taken prior to surgery , while the post - chemotherapy sample was taken five months after the therapy was completed . the biological fluid examined was serum . haptoglobin - 1 precursor and ca 125 values were determined in serum samples taken before and after each cycle of therapy . the expression of haptoglobin - 1 precursor before and after the chemotherapy is shown in fig9 a and 9 b . it can be seen that the level of expression of haptoglobin - 1 precursor decreased after chemotherapy relative to the level before chemotherapy . before surgery the level of ca 125 in serum from this patient was 376 u / ml . after completion of the chemotherapy the level of ca 125 had reduced to 16 u / ml . hence the decrease in the level of haptoglobin - 1 precursor in the biological fluid after chemotherapy treatment correlated with the decrease in the level of ca 125 in the biological fluid after chemotherapy . evaluation of haptoglobin - 1 precursor concentration and its isoforms in biological fluids the efficacy of ovarian cancer treatment , recurrence of disease following treatment , and the early detection of the onset of ovarian cancer is evaluated by quantifying the concentration of haptoglobin - 1 precursor and its isoforms in biological fluids by ( i ) direct or indirect sandwich elisa using either polyclonal or monoclonal antibodies that target intermediate and βchain epitopes ( ii ) fluorescent bead - based immunoassasy ( luminx technology ) and / or ( iii ) magnetic bead - based immunoassay and mass spectrometry analysis . the assay of haptoglobin - 1 precursor is performed in conjunction with the determination of other analytes associated with ovarian cancer using methods known in the art , including but not limited to elisa assays for ca125 ( mackay and creasman , 1995 ), ilk ( pct / au03 / 01058 ), tadg - 12 ( underwood l j , 2000 ), serotransferrin ( kawakami et al , 1999 ), neutrophil gelatinase associated lipocalin ( kjeldsen et al , 1994 ), soluble cd163 ( baeton et al , 2003 ) and gc - globulin ( jorgensen et al , 2004 ). as shown in table 3 , the level of expression of haptoglobin - 1 precursor can be used in conjunction with other markers in order to improve the sensitivity and specificity of the test . the lack of suppression of haptoglobin - 1 precursor in patient 2 after chemotherapy correlates with an increase in ca 125 concentration . this result may indicate the development of resistance to the chemotherapeutic agent , and is being further investigated . our findings show that serum concentrations of haptoglobin - 1 precursor are significantly increased in early stage ovarian cancer patients . without wishing to limit any proposed mechanism , we believe that enhanced hepatic synthesis of haptoglobin precursor may occur due to an acute phase response in ovarian cancer patients , resulting in elevated concentrations of serum haptoglobin precursor . we have demonstrated a semi - quantitative correlation between the level of haptoglobin precursor expression and the grade of the cancer . we envisage that a quantitative correlation can readily be established using quantitative assay methods such as immunoassay . we consider that haptoglobin - 1 precursor represents a new and useful biomarker for ovarian cancer diagnosis . the lack of immunoreactive haptoglobin - 1 precursor expression in normal epithelium and the increased expression of immunoreactive haptoglobin - 1 precursor in advanced stage tumours suggests that haptoglobin - l precursor is critical for ovarian cancer progression . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . beutler , e ., gelbart , t ., and lee , p . haptoglobin polymorphism and iron homeostasis , clin chem . 48 : 2232 - 5 ., 2002 . diamandis , e . p ., yousef , g . m ., soosaipillai , a . r ., and bunting , p . 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