Patent Application: US-75263500-A

Abstract:
the invention relates to the field of cancer diagnosis and the application of diagnostic techniques in pathology and hematology . specifically , the invention relates to flow cytometric techniques for the detection of chromosomal aberrations and the detection of tumor specific gene products exclusively expressed by tumor cells containing said chromosomal aberrations . the invention provides a method to detect chromosomal aberrations in a biological sample via the exclusive detection of tumor - specific gene - product using at least two different probes directed against the gene - product .

Description:
the experimental part describes more in detail the invention relating to the field of leukemia , but can in no way be seen as limiting the invention . the reciprocal translocationt ( 9 ; 22 )( q34 ; q11 ), observed in chronic myeloid leukemia ( cml ), acute lymphoblastic leukemia ( all ), and acute myeloid leukemia ( aml ), results from fusion between two genes : bcr and abl . depending on the localization of the breakpoint in the bcr gene , different tumor specific bcr - abl genes are generated . these bcr - abl genes are transcribed and translated in tumor - specific bcr - abl mrna and tumor specific bcr - abl proteins , respectively . hence , different diagnostic targets are available , each allowing specific diagnosis of t ( 9 ; 22 )( q34 ; q11 ) positive leukemia . while conventional cytogenetics relies on detection of the characteristic chromosomal aberration ( i . e ., the philadelphia chromosome : a minute chromosome 22 ), other techniques are used to specifically detect the bcr - abl fusion - gene ( e . g ., fluorescent in situ hybridization ) or the bcr - abl fusion mrna ( e . g ., reverse transcriptase polymerase chain reaction ). although all of the aforementioned techniques are well established as diagnostic techniques , none of these techniques can be easily performed on a routine and short - term basis . yet , especially in all , presence of the philadelphia ( ph ) chromosome is associated with a poor prognosis . to improve the poor prognostic outcome , ph positive alls require early identification to permit intensive induction regimens or alternative treatment protocols . a new diagnostic technique is presented that is based on the exclusive detection of tumor - specific fusion - proteins . this technique is designed for identification of cancers such as ph positive leukemias at first diagnosis in a rapid and simple fashion . the ph chromosome was the first karyotypic aberration found to be tumor - related . to date , the ph chromosome is identified in various hematopoietic disorders ; e . g ., cml , all , and aml , in both adults and children . the ph chromosome is generated by the reciprocal translocation between the long arms of chromosome 9 and 22 : t ( 9 ; 22 )( q34 ; q11 ) and involves the abl gene on chromosome 9 and the bcr gene on chromosome 22 . both genes are interrupted and rearranged ; resulting in a tumor - specific bcr - abl fusion - gene on chromosome 22q - and a abl - bcr fusion - gene on chromosome 9 +. while reports on the abl - bcr fusion - gene are still limited , bcr - abl fusion - genes have been extensively studied over the past two decades . depending on the chromosomal localization of the breakpoints , different types of bcr - abl fusion - genes have been identified . it has been demonstrated that breakpoints in the bcr gene are clustered within two regions : the major breakpoint cluster region ( m - bcr ), comprising five exons termed b1 to b5 ; and a minor breakpoint cluster region ( m - bcr ), located 5 ′ of the m - bcr in the bcr - gene . in contrast , breakpoints in the abl gene are scattered over long distances and mostly occur 5 ′ of exon a2 . in both ph + cml patients as well as ph + all patients breakpoints in the m - bcr are evenly distributed : either located between exon b2 and b3 or located between exon b3 and b4 . breakpoints in ph + all are , however , in majority ( app . 70 %) found within the m - bcr , localized in an intron between exon e1 and e2 . because breakpoints are scattered over long distances ( especially in the abl gene ), different fusion - point introns are generated within bcr - abl genes . although these fusion - point introns are highly variable between ph + patients when considering the bcr - abl gene &# 39 ; s fusion - point intron &# 39 ; s length and nucleotide sequence , fusion - points of bcr - abl transcripts are highly consistent . thus , depending on the original bcr - abl gene rearrangement , a single kind of bcr - abl mrna is usually detected : varying from a 7 kb mrna comprising an e1a2 junction to a 8 . 5 bcr - abl mrna that either comprises a b2a2 or b3a2 junction . as the translational reading frame of bcr - abl mrnas is maintained , ph + leukemia cells express unique bcr - abl proteins . while ph chromosomes are almost invariably present in cml cases , ph chromosomes are less often detected in leukemia cells from patients suffering from aml or all . still , 5 % of aml cases , 25 % to 30 % of adults with all and 3 % to 5 % of children with all are diagnosed as ph +. reflected by a high rate of treatment failure and mortality in ph + leukemias , in both adults and children , ph chromosomes are hallmarked as significant risk - factors considering treatment failure . the importance of identifying risk - factors , such as the ph chromosome , is beyond doubt . current treatment protocols may be improved by identification of the t ( 9 ; 22 )( q11 ; q34 ) at an early time - point of the disease . at present , ph + leukemias are identified by a number of techniques , either detecting the aberrant chromosome , the gene , the mrna or the aberrant protein . yet , each of these techniques is characterized by typical specifications and limitations which should be considered before one attempts to diagnose t ( 9 ; 22 )( q34 ; q11 ) positive leukemias specifically . herein is described : an assay developed to discriminate between ph + leukemias and ph — leukemias at first diagnosis in a relatively rapid and simple fashion . the underlying principle of the assay is based on detection of tumor - specific proteins by antibodies specifically reactive with fragments corresponding to the bcr - abl fusion - proteins and to fractions of the original , non - fused bcr and abl proteins . cell lines : six ph + cell lines were used to examine the specificity of both the sepharose - western blotting procedure as well as the bcr - abl dipstick assay : lama - 84 and k562 , kcl - 22 and bv - 173 , and tom - 1 and all / mik . all cell lines were cultured in rpmi - 1640 supplemented with 10 % fetal calf serum . leukemic cell samples : two leukemic cryopreserved peripheral blood samples from leukemic patients at diagnosis were used to examine the specificity of the bcr - abl dipstick assay . clinical and laboratory data of these patients have been described previously : one patient suffered from a ph negative cml , with rearranged b2a2 bcr - abl genes , the other suffered from a ph positive precursor b - all , with rearranged e1a2 bcr - abl genes . all antibodies used were protein g purified and categorized as : catching antibodies : monoclonal antibody ( moab ) 7c6 ( a generous gift from dr . s . dhut ), directed towards the b2 - epitope present in b2a2p210 bcr - abl , b3a2p210 bcr - abl , p160 bcr and p130 bcr ; moab er - fp1 , directed towards the e1a2 fusion - point in e1a2p190 bcr - abl and ; moab yae ( santa cruz biotechn ., santa cruz , calif ., usa ) directed towards the amino - terminus of e2a proteins . detecting antibodies : moab 8e9 ( a generous gift from dr . j . wang ), directed towards the sh2 domain present in e1a2p190 bcr - abl , b2a2p190 bcr - abl , b3a2p190 bcr - abl and p145 abl and ; moab g98 - 271 . 1 . 3 ( a generous gift from dr . g . bain ) directed towards the carboxyl terminus of e2a proteins . both moab 8e9 and moab g98 - 271 . 1 . 3 were biotinylated . cells were washed twice with ice - cold phosphate buffered saline ( pbs ) and lysed in ice - cold lysis buffer ( 1 % triton x - 100 , 0 . 05 % sodium dodecyl sulphate ( sds ), 150 mm nacl , 5 mm edta in 10 mm sodium phosphate , ph 7 . 0 ), supplemented with 40 μl phenyl methyl sulfonyl fluoride ( pmsf : 100 mm in 2 - isopropanol ) at a concentration of 1 × 10 7 cells / ml for 15 min . after the lysates were centrifuged in an eppendorf centrifuge to remove insoluble material ( 5 min 4 ° c . ), supernatants were split into equal volumes representing 10 7 cells . sepharose - western blotting was performed by adding either 10 μg moab 7c6 or 2 μg moab er - fp1 to the supernatant of lysed cells . antigen - antibody reaction was allowed for two hours on a rotation device at 4 ° c . next , 40 μl of an 80 % ( v / v ) suspension of gammabind g sepharose beads ( pharmacia biotech ab , uppsala , sweden ) were added . after 30 min , beads were collected and washed three times in lysis - buffer without sds . beads were boiled for 5 min in 60 μl sample buffer ( 60 mm tris - hcl , ph 6 . 8 , 10 % glycerol , 10 mm edta , 2 % sds , 2 % b - mercaptoethanol and 0 , 03 % bromophenol blue . protein samples were subjected to 6 % sds - page and transferred ( mini protean ; bio rad , richmond , calif ., usa ) to nitrocellulose ( 0 . 45 μm pore size ; schleicher & amp ; schuell , dassel , germany ). nitrocellulose sheets were blocked in 5 % non - fat dry milk powder ( protifar , nutricia , the netherlands ) in pbs supplemented with 0 . 05 % tween - 20 ( 5 % mpbs ). next , sheets were incubated for two hours at room temperature in the presence of biotinylated moab 8e9 ( 2 μg / ml ) in 1 % mpbs . following three washes with pbs supplemented with 0 . 05 % tween - 20 , alkaline phosphatase conjugated to streptavidin ( south . biotechn . ass ., birmingham , ala ., usa ) was added to a 1 : 1500 dilution and incubation was allowed to proceed for one hour . the blot was washed twice with pbs supplemented with 0 . 05 % tween - 20 and finally with 0 . 15 m veronal acetate buffer , ph 9 . 6 . for visualization of antibody - antigen complexes , we used the alkaline phosphatase substrate nitro blue tetrazolium / 5 - bromo - 4 - chloroindoxyl phosphate ( nbt / bcip ; sigma , st . louis , mo ., usa ) as previously described . each catching antibody was applied as a single small spot to a (± 2 cm × 0 . 5 cm ) nitrocellulose ( 0 . 45 μm pore size ) strip and air dried . each spot contained either 2 μg of moab 7c6 , 1 μg moab er - fp1 or 1 μg moab yae . next , these nitrocellulose strips , called ‘ dipsticks ’, were rinsed in pbs supplemented with 0 . 05 % tween - 20 and subsequently blocked in 5 % mpbs ( 1 h , rt ). at this point , dipsticks can be air dried and stored in an airtight container at 4 ° c . until further use . supernatants of cellular lysates ( processed and described in the first paragraph of the above section ), representing 10 7 cells , were added to the dipsticks . antigen - antibody complex formation was allowed to proceed overnight at 4 ° c . on a rotation device . next , dipsticks were rinsed three times in pbs supplemented with 0 . 05 % tween - 20 and bound antigens were detected by incubating the dipstick with a mixture of biotinylated moab 8e9 ( 2 μg / ml ) and biotinylated moab g98 - 271 . 1 . 3 ( 2 μg / ml ), diluted in 1 % mpbs . from this point on , dipsticks were further processed as described in the materials and method section of the sepharose - western blotting procedure . to determine whether the tumor - specific bcr - abl fusion - proteins can be exclusively recognized by immunological methods , we developed a sepharose - western blotting procedure . a sepharose - western blotting procedure is a combination of an immunoprecipitation reaction with a catching antibody , followed by a western blotting procedure with a detecting antibody . moabs 7c6 or er - fp1 were used as catching antibody , precipitating proteins from cellular lysates of lama - 84 and kcl - 22 , or tom - 1 cells , respectively . following immunoblotting , precipitated proteins were detected by the use of biotinylated moab 8e9 as detecting antibodies and alkaline phosphatase conjugated to streptavidin . these sepharose - western blotting experiments with 7c6 / 8e9 antibody combinations , as well as those using antibody combinations er - fp1 / 8e9 enabled exclusive detection of bcr - abl proteins . the antibody er - fp1 combination detects e1a2p190 bcr - abl proteins that are not detected by the 7c6 / 8e9 antibody combination . yet , the combination of 7c6 / 8e9 specifically detects b2a2p210 bcr - abl and b3a2p210 bcr - abl proteins , both of which are not recognized by er - fp1 antibodies . in conclusion , our sepharose - western blotting data verify that tumor - specific bcr - abl fusion - proteins are exclusively identified by the appropriate choice and mix of antibodies . we next investigated whether the sepharose - western blotting procedure could be simplified . by using the same sets of antibodies as were used in the sepharose - western blotting experiments , an alternative bcr - abl detection system , termed the bcr - abl dipstick , was investigated for its capability identifying bcr - abl proteins exclusively . the bcr - abl dipstick is made of nitrocellulose strips on which three different antibodies are immobilized : 1 ) moab7c6 , 2 ) moab er - fp1 and 3 ) moab yae . to investigate whether the bcr - abl dipstick can be used for specific identification of bcr - abl proteins , bcr - abl dipsticks were either incubated with cellular lysates from : 1 ) lama - 84 , 2 ) kcl - 22 or 3 ) tom - 1 cells . fusion - proteins that had been caught by immobilized antibodies were detected by subsequent incubation with a mixture of biotinylated moab 8e9 ( 8e9 - bio , recognizing the carboxyl terminus of both abl and bcr - abl proteins ) and biotinylated moab g98 - 272 . 1 . 3 . ( recognizing the carboxyl terminus of e2a - proteins ) followed by alkaline phosphatase conjugated to streptavidin . incubating a bcr - abl dipstick with either cellular lysates from lama - 84 or cellular lysates from kcl - 22 , results , upon successive incubation with biotinylated antibodies ( i . e . 8 e 9 - bio and g98 . 271 . 1 . 3 - bio ), streptavidin - ap and its substrate , in visible dots located at the moab 7c6 antibody spot . incubating a bcr - abl dipstick with cellular lysates from tom - 1 , results , upon subsequent incubation with the aforementioned molecules , in a visible dot located at the er - fp - 1 antibody spot . considering the sepharose - western blotting data described above , these dots represent bound bcr - abl proteins . together , these data demonstrate that the bcr - abl dipstick assay can be applied for the exclusive detection of tumor - specific bcr - abl proteins . at this point , the bcr - abl dipstick assay specifically detects bcr - abl proteins in cellular lysates made from cell lines . next , we investigated whether the bcr - abl dipstick assay can be applied for specific diagnosis of bcr - abl positive leukemias . two cryopreserved , ficoll - enriched blood samples from patient a and patient b , respectively , with previously diagnosed bcr - abl positive leukemias , were lysed and investigated by both the bcr - abl dipstick assay as well as the sepharose - western blotting procedure . the blood samples from patient a and patient b represent a ph — cml with cryptic rearranged b3a2bcr - abl genes and a ph + all with rearranged e1a2bcr - abl genes , respectively . both samples scored positive for the presence of bcr - abl fusion - protein . the presence of the ph chromosome in leukemic cells is associated with poor prognosis . especially in all , it is important to distinguish ph + leukemias from ph — leukemias , as presence of the ph chromosome identifies a large group of patients facing an insecure future . yet , this poor therapeutic outcome may be improved by an early start with more aggressive induction therapies . therefore , sensitive and reliable diagnostic methods , identifying the ph chromosome or its products at an early time - point of the disease , are extremely important in all diagnosis . at present , conventional cytogenetic analysis is the method of choice for identifying various chromosomal abnormalities in all . however , the results obtained by cytogenetic analysis are not always reliable since results largely depend on the number of metaphases investigated . only institutions with special experience in all cytogenetics achieve successful karyotype analysis in almost every patient . even then , some cryptic bcr - abl rearrangements escape detection by conventional cytogenetic analysis . contrary to conventional cytogenetics , fluorescent in situ hybridization ( fish ) techniques are not limited to the laborious analysis of metaphases . by applying probes directed against bcr and abl genes , each labeled with a different fluorochrome , ph + interphase cells can be identified . yet , depending on the co - localization of the two hybridization signals to one spot , its sensitivity is limited , because artefactual co - localization in non - malignant , normal cells may be observed . the polymerase chain reaction ( pcr ) is at present the most sensitive method for detecting genetic abnormalities . in fact , molecular analysis frequently detects aberrations that are not observed karyotypically . however , as breakpoints are scattered over long distances in the tumor - specific fusion - point introns , the pcr procedure is only applicable after reverse transcription of bcr - abl messenger rna . being very sensitive , strict precautions are required to prevent false positive ( due to cross - contamination ) and false negative ( due to premature mrna degradation ) results . here , we describe the development of a new , simple and rapid technique based on detection of two distinct antigenic sites on the bcr - abl fusion - protein . the combined specificity of at least two different antibodies allows for exclusive detection of bcr - abl proteins within 24 hours . our assumptions concerning exclusive immunological detection of bcr - abl proteins by the proper combination of antibodies proved correct as they were first tested in sepharose - western blotting experiments . these experiments demonstrate that b3a2bcr - abl and b2a2bcr - abl proteins are specifically identified by the moan 7c6 / 8e9 - bio combination , while e1a2bcr - abl proteins are specifically identified by the moab er - fp1 / 8e9 - bio combination . we next investigated whether the sepharose - western blotting procedure could even be more simplified . the resulting bcr - abl dipstick , a small nitrocellulose strip on which three different antibodies are immobilized , was examined for both specificity and sensitivity by using different ph + cell lines . the specificity was confirmed by the analysis of ph + cell lines : each expressing a different type of bcr - abl protein . these results are consistent and were also observed upon testing other ph + cell lines such as k562 , bv173 and mik - all . the results show that this assay can act as an alternative screening method for detecting bcr - abl positive leukemias at first diagnosis . dipstick analysis of two leukemic cell samples with previously reported rearranged bcr - abl genes , showed that initial diagnosis of bcr - abl positive leukemias is indeed feasible . moreover , its surplus value considering conventional cytogenetics is demonstrated by the analysis of patient a . even though this patient suffered from ph negative cml with cryptic rearranged bcr - abl genes , bcr - abl proteins were readily identified upon using the bcr - abl dipstick assay . chromosomal aberrations , which are frequently present in malignant cells , can result in altered expression of genes ( e . g ., bcl2 protein overexpression in lymphomas with t ( 14 ; 18 ) in which the bcl2 gene is linked to the igh promoter ) or in aberrant fusion genes ( e . g ., t ( 1 ; 19 ) in which a part of the e2a gene is linked to a part of the pbx1 ). fusion genes may result in the presence of fusion gene transcripts , which may subsequently be translated into fusion proteins ( fig1 ). chromosomal aberrations with fusion genes occur in several types of malignancies . for example , t ( 9 ; 22 ) is found in & gt ; 95 % of chronic myeloid leukemias ( cml ) and in 25 to 40 % of adult precursor - b - acute lymphoblastic leukemias ( all ), whereas t ( 12 ; 21 ) with the tel - aml1 fusion gene is found in approximately 25 % of childhood precursor - b - all . detection of these chromosomal aberrations is of diagnostic relevance , because specific chromosomal aberrations with fusion genes can be used as pcr prognostic factors in several types of cancer , e . g ., the presence of t ( 4 ; 11 ) in infant leukemia is associated with poor outcome . in addition , chromosomal aberrations can be used as targets for monitoring the level of residual disease during and after therapy , thereby providing insight into the effectiveness of treatment . therefore , detection of chromosomal aberrations with fusion genes is relevant for making the appropriate diagnosis , for classification , and for evaluating the effectiveness of treatment . chromosomal aberrations with fusion genes can be detected at several levels and by several techniques : at the dna level ( e . g ., by fish ), at the mrna level ( e . g ., by rt - pcr analysis ), and at the protein level ( e . g ., by elisa ). if chromosomal aberrations with fusion genes are analyzed at the protein level , an antibody sandwich technique is used . this technique is based on a catching antibody , recognizing part of the fusion protein coded for by gene a , and a detection antibody , recognizing part of the fusion protein coded for by gene b ( fig1 ). in most cases , the catching antibody is linked to a carrier , such as a membrane ( e . g ., dipstick assay ) or a well plate ( e . g ., elisa ). although these methods in general are rapid and efficient , they are hard to incorporate into a routine hematological laboratory , because these techniques and the corresponding equipment are not readably available in such laboratories . however , flow cytometry is widely used in hematological laboratories and therefore a flow cytometric assay for detection of chromosomal aberrations would be preferred . here we propose an assay for the analysis of chromosomal aberrations , using a detection method that is based on beads , which have the appropriate characteristics for flow cytometry . 1 . a catching antibody , recognizing part of the fusion gene protein coded for by gene a , is coupled to a particle such as a bead ; 2 . a cell lysate ( e . g ., from leukemic cells or other malignant cells ) is added to the antibody - coated beads ; 3 . after extensive washing of the beads , the bead mixture is incubated with a detection antibody , recognizing a different part of the fusion gene protein coded for by gene b ; this antibody is conjugated with a fluorochrome or any other visualization method , which is suitable for flow cytometric detection ; 4 . after extensive washing , the beads are analyzed by flow cytometry . for reasons of efficacy , it will be convenient to investigate the occurrence of different fusion gene proteins simultaneously in one tube . this is not because a particular malignancy will have more than one fusion gene protein , but because it is convenient to have a single test tube for detection of several well - established fusion gene proteins within one disease category , e . g ., acute myeloid leukemia ( aml ) or precursor - b - all ( table 1 and fig2 ). for discrimination between different types of fusion proteins , different beads are used . these beads may differ in size , conjugated fluorochrome , intensity of conjugated fluorochrome , or other bead characteristics ( fig2 ). in several types of malignancies , the same target gene might be involved in different variant translocations , resulting in fusion to different partner genes ( table 2 ). consequently , the same target gene ( e . g ., mll or ews ) produces different fusion proteins , depending on the type of variant translocation . the variant fusion proteins can be detected in a single tube assay via usage of differently labeled detection antibodies . for example the different types of fusion proteins from the variant translocations involving the mll gene in infant precursor - b - all ( table 2 ) can be detected by use of differently conjugated af4 , af9 , and enl antibodies ( fig3 ). finally , some fusion proteins can occur in variant forms dependent on the breakpoint region , such as in t ( 9 ; 22 ), t ( 15 ; 17 ), and inv ( 16 ). for instance the bcr gene , involved in t ( 9 ; 22 ), is known to have three breakpoint regions : major breakpoint cluster region ( m - bcr ), minor breakpoint cluster region ( m - bcr ), and micro breakpoint cluster region ( m - bcr ) ( table 3 and fig4 ). depending on the breakpoint region , several bcr exons may be present or absent in the coding regions of the different types of bcr - abl fusion proteins . discrimination between the three variant bcr - abl fusion proteins is possible via antibodies , which recognize the different bcr domains , which are present in one variant form but absent in the other ( see , table 3 and fig4 ). the herein described bead - based sandwich antibody technique allows easy and rapid flow cytometric detection of different types of fusion proteins in a single tube assay by using different bead - bound catching antibodies against one part of the different fusion proteins and the relevant corresponding detection antibodies against the other part of the fusion proteins . based on different flow cytometric characteristics of the beads ( e . g ., size , fluorochrome color , intensity of fluorochrome staining , or side scatter characteristics ), multiple fusion proteins can be specifically detected in the same assay ( fig2 - 4 ). this also includes the detection of fusion proteins from various variant translocations of the same target gene ( table 2 and fig3 ) as well as fusion proteins from translocations with variant breakpoints ( table 3 and fig4 ). the herein presented bead - based flow cytometric technique for detection of fusion proteins is applicable on cell lysates of any malignancy with a chromosomal aberration that results in a fusion gene with the expression of the corresponding fusion protein . carson r , vignali d . simultaneous quantitation of fifteen cytokines using a multiplexed flow cytometric assay . j immunol methods 1999 ; 227 : 41 - 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