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WO2009153175A1 - Procédés et moyens de diagnostic du cancer du pancréas - Google Patents

Procédés et moyens de diagnostic du cancer du pancréas Download PDF

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Publication number
WO2009153175A1
WO2009153175A1 PCT/EP2009/056951 EP2009056951W WO2009153175A1 WO 2009153175 A1 WO2009153175 A1 WO 2009153175A1 EP 2009056951 W EP2009056951 W EP 2009056951W WO 2009153175 A1 WO2009153175 A1 WO 2009153175A1
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WIPO (PCT)
Prior art keywords
amount
subject
pancreatic cancer
sic3b
sample
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PCT/EP2009/056951
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English (en)
Inventor
Angela MÄRTEN
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Universitätsklinikum Heidelberg
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Publication of WO2009153175A1 publication Critical patent/WO2009153175A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4716—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention pertains to the field of cancer diagnosis. Specifically, it relates to a method for diagnosing pancreatic cancer in a subject comprising the steps of determining the amount of siC3b in a sample of a subject and comparing said amount to a reference amount, whereby pancreatic cancer is to be diagnosed.
  • the present invention also contemplates a method of determining whether a subject should be closely monitored for pancreatic cancer and a method for determining whether an anti-cancer therapy applied to a subject after treatment of pancreatic cancer is beneficial. Contemplated are, furthermore, diagnostic devices and kits for carrying out said methods.
  • PDAC pancreatic ductal adenocarcinoma
  • the complement system provides a rapid and efficient means to protect the host from invasive microorganisms and has anti-tumor properties. Under physiological conditions, an uncontrolled activation of complement is prevented by a vast array of regulatory proteins, either circulating in the plasma, or expressed on the cell surface (Morgan 1999, Crit Rev. Immunol. 19: 173-98). In cancer patients, complement activation with subsequent deposition of complement components on tumor tissue has been demonstrated (Ytting 2004, Scand J Gastroenterol 39:674-9; Ajona 2004, Cancer Res, 64:6310-8; Gelderman 2003, MoI Immunol 40:13-23; Baatrup 1994, Eur J Surg 160:503-10; Niculescu 1992, Am J Pathol 140: 1039-43).
  • CD35 complement receptor type 1 , CRl
  • CD55 decay accelerating factor, DAF
  • CD46 membrane cofactor protein, MCP
  • CD59 Protectedin
  • Membrane complement regulatory molecules are found on normal and malignant cells, and their level of expression, even within the same tissues, is very heterogeneous.
  • mCRP membrane complement regulatory molecules
  • CA 19.9 is a tumor-associated antigen, originally isolated from a human colon cancer cell line. It is present on gangliosides in tissues, but is carried by glycoproteins in serum. The oligosaccharide, bearing the CA 19.9 antigen, is related to sialylated Lewis A blood group antigen. Lewis A antigen must be present before CA 19.9 can be expressed.
  • CA 19.9 is synthesized by normal cells in pancreatic and bile ducts, gastric and colonic mucosa, bronchial and salivary glands, endometrium, and prostate.
  • CA 19.9 is inferior for screening of pancreatic cancer in asymptomatic patients since its sensitivity for early cancer is low. Serum levels are elevated in less than 30% of patients with stage 1 cancers. Moreover, elevated CA 19.9 levels are not specific for pancreatic cancer, but are elevated in other benign and malignant disorders. Therefore, there is still a need for a more reliable biomarker for diagnosing pancreatic cancer. In light of the severe consequences of the disease and the unspecific clinical symptoms at the beginning of the disease, such a biomarker could strengthen diagnostic and therapeutic approaches against pancreatic cancer. Accordingly, it is the technical problem underlying this invention to provide means and methods complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.
  • the present invention relates to a method for diagnosing pancreatic cancer in a subject comprising the steps of a) determining the amount of siC3b in a sample of a subject; and b) comparing said amount to a reference amount, whereby pancreatic cancer is to be diagnosed.
  • the method of the present invention is an in vitro method. Moreover, it may comprise steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pre-treatments or evaluation of the results obtained by the method.
  • the method may be carried out manually or assisted by automation.
  • step (a) and/or (b) may in total or in part be assisted by automation, e.g., by a suitable robotic and sensory equipment for the determination in step (a) or a computer-implemented comparison in step (b).
  • the term "diagnosing” as used herein means assessing whether a subject suffers from pancreatic cancer. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for all (i.e. 100%) of the subjects to be identified. The term, however, requires that a statistically significant portion of subjects can be identified (e.g. a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g. , determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test etc.. Details are found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99 %.
  • the p-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. More preferably, at least 60%, at least 70%, at least 80% or at least 90% of the subjects of a population can be properly identified by the method of the present invention. Diagnosing according to the present invention includes applications of the method in monitoring, confirmation, and subclassification of the relevant disease or its symptoms.
  • pancreatic cancer refers to cancer which is derived from pancreatic cells.
  • pancreatic cancer as used herein is pancreatic carcinoma, most preferably, pancreatic ductal adenocarcinoma, PDAC.
  • PDAC pancreatic ductal adenocarcinoma
  • subject as used herein relates to animals, preferably mammals, and, more preferably, humans.
  • the method of the present invention will be applied for subjects which previously suffered from pancreatic cancer or to subjects and which have been treated by, e.g., resection of the pancreatic cancer, or to subjects which are at increased risk of developing pancreatic cancer, more preferably, subjects suffering from chronic pancreatitis, subjects with a familiar background (i.e. subjects from families where family members suffered already from pancreatic cancer) or subjects with genetic mutations influencing pancreatic cancer, e.g., Peutz-Jeghers syndrome.
  • siC3b refers to a fragment of the complement factor C3b.
  • the complement factor C3b is obtained after cleavage of the C3 complement factor into C3a and C3b.
  • C3b can be subsequently cleaved into its inactive form iC3b.
  • Said iC3b can be detected in the fluid phase as siC3b.
  • siC3b as used herein preferably, refers to human siC3b.
  • the structure of human siC3b is well known in the art and, preferably, disclosed in Sarrias MR, Franchini S, Canziani G, Argyropoulos E, Moore WT, Sahu A, Lambris JD.
  • the term encompasses also variants of the aforementioned specific siC3b. Such variants have at least the same essential biological and immunological properties as the specific siC3b. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification, e.g., by ELISA Assays using polyclonal or monoclonal antibodies specifically recognizing the said siC3b.
  • a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and/or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the amino sequence of the specific siC3b.
  • Variants may be allelic variants or any other species specific homologs, paralogs, or orthologs.
  • the variants referred to herein include fragments of the specif ⁇ c siC3b or the aforementioned types of variants as long as these fragments have the essential immunological and biological properties as referred to above. Such fragments may be, e.g., further degradation products of the siC3b. Further included are variants which differ due to posttranslational modifications.
  • sample refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ.
  • Samples of body fluids can be obtained by well known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum.
  • Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy.
  • Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting.
  • cell-, tissue- or organ samples are obtained from those cells, tissues or organs which express or produce the peptides referred to herein.
  • Determining the amount of the polypeptides referred to in this specification relates to measuring the amount or concentration, preferably semi-quantitatively or quantitatively. Measuring can be done directly or indirectly.
  • Direct measuring relates to measuring the amount or concentration of the polypeptide based on a signal which is obtained from the polypeptide itself and the intensity of which directly correlates with the number of molecules of the polypeptide present in the sample. Such a signal - sometimes referred to herein as intensity signal -may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the polypeptide.
  • Indirect measuring includes measuring of a signal obtained from a secondary component (i.e. a component not being the polypeptide itself) or a biological read out system, e.g., measurable cellular responses, ligands, labels, or enzymatic reaction products.
  • determining the amount of a polypeptide can be achieved by all known means for determining the amount of a polypeptide in a sample.
  • Said means comprise immunoassay devices and methods which may utilize labeled molecules in various sandwich, competition, or other assay formats. Said assays will develop a signal which is indicative for the presence or absence of the polypeptide.
  • the signal strength can, preferably, be correlated directly or indirectly (e.g. reverse- proportional) to the amount of polypeptide present in a sample.
  • Further suitable methods comprise measuring a physical or chemical property specific for the polypeptide such as its precise molecular mass or NMR spectrum. Said methods comprise, preferably, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass- spectrometers, NMR- analyzers, or chromatography devices. Further, methods include micro-plate ELISA-based methods, fully-automated or robotic immunoassays, CBA (an enzymatic Cobalt Binding Assay), and latex agglutination assays. A preferred assay for determining the amout of siC3b is the iC3b ELISA from Quidel Corp, USA.
  • determining the amount of a polypeptide comprises the steps of (a) contacting a cell capable of eliciting a cellular response the intensity of which is indicative of the amount of the polypeptide with the said polypeptide for an adequate period of time, (b) measuring the cellular response.
  • the sample or processed sample is, preferably, added to a cell culture and an internal or external cellular response is measured.
  • the cellular response may include the measurable expression of a reporter gene or the secretion of a substance, e.g. a peptide, polypeptide, or a small molecule.
  • the expression or substance shall generate an intensity signal which correlates to the amount of the polypeptide.
  • determining the amount of a polypeptide comprises the step of measuring a specific intensity signal obtainable from the polypeptide in the sample.
  • a specific intensity signal may be the signal intensity observed at a mass to charge (m/z) variable specific for the polypeptide observed in mass spectra or a NMR spectrum specific for the polypeptide.
  • Determining the amount of a polypeptide may, preferably, comprises the steps of (a) contacting the polypeptide with a specific ligand, (b) (optionally) removing non-bound ligand, (c) measuring the amount of bound ligand.
  • the bound ligand will generate an intensity signal.
  • Binding according to the present invention includes both covalent and non-covalent binding.
  • a ligand according to the present invention can be any compound, e.g., a peptide, polypeptide, nucleic acid, or small molecule, binding to the polypeptide described herein.
  • Preferred ligands include antibodies, nucleic acids, peptides or polypeptides such as receptors or binding partners for the polypeptide and fragments thereof comprising the binding domains for the peptides, and aptamers, e.g. nucleic acid or peptide aptamers.
  • Methods to prepare such ligands are well-known in the art. For example, identification and production of suitable antibodies or aptamers is also offered by commercial suppliers. The person skilled in the art is familiar with methods to develop derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into the nucleic acids, peptides or polypeptides. These derivatives can then be tested for binding according to screening procedures known in the art, e.g.
  • Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F(ab) 2 fragments that are capable of binding antigen or hapten.
  • the present invention also includes single chain antibodies and humanized hybrid antibodies wherein amino acid sequences of a non- human donor antibody exhibiting a desired antigen-specificity are combined with sequences of a human acceptor antibody.
  • the donor sequences will usually include at least the antigen-binding amino acid residues of the donor but may comprise other structurally and/or functionally relevant amino acid residues of the donor antibody as well.
  • Such hybrids can be prepared by several methods well known in the art.
  • the ligand or agent binds specifically to the polypeptide.
  • Specific binding means that the ligand or agent should not bind substantially to ("cross-react" with) another peptide, polypeptide or substance present in the sample to be analyzed.
  • the specifically bound polypeptide should be bound with at least 3 times higher, more preferably at least 10 times higher and even more preferably at least 50 times higher affinity than any other relevant peptide or polypeptide.
  • Non-specific binding may be tolerable, if it can still be distinguished and measured unequivocally, e.g. according to its size on a Western Blot, or by its relatively higher abundance in the sample.
  • Binding of the ligand can be measured by any method known in the art. Preferably, said method is semiquantitative or quantitative. Suitable methods are described in the following. First, binding of a ligand may be measured directly, e.g. by NMR or surface plasmon resonance.
  • an enzymatic reaction product may be measured (e.g. the amount of a protease can be measured by measuring the amount of cleaved substrate, e.g. on a Western Blot).
  • the ligand may exhibit enzymatic properties itself and the "ligand/ polypeptide" complex or the ligand which was bound by the polypeptide, respectively, may be contacted with a suitable substrate allowing detection by the generation of an intensity signal.
  • the amount of substrate is saturating.
  • the substrate may also be labeled with a detectable lable prior to the reaction.
  • the sample is contacted with the substrate for an adequate period of time.
  • An adequate period of time refers to the time necessary for a detectable, preferably measurable, amount of product to be produced. Instead of measuring the amount of product, the time necessary for appearance of a given (e.g. detectable) amount of product can be measured.
  • the ligand may be coupled covalently or non-covalently to a label allowing detection and measurement of the ligand. Labeling may be done by direct or indirect methods. Direct labeling involves coupling of the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) of a secondary ligand to the first ligand.
  • the secondary ligand should specifically bind to the first ligand.
  • Said secondary ligand may be coupled with a suitable label and/or be the target (receptor) of tertiary ligand binding to the secondary ligand.
  • the use of secondary, tertiary or even higher order ligands is often used to increase the signal.
  • Suitable secondary and higher order ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.).
  • the ligand or substrate may also be "tagged" with one or more tags as known in the art. Such tags may then be targets for higher order ligands.
  • Suitable tags include biotin, digoxygenin, His-Tag, Glutathion-S- Transferase, FLAG, GFP, myc-tag, influenza A virus haemagglutinin (HA), maltose binding protein, and the like.
  • the tag is preferably at the N-terminus and/or C-terminus.
  • Suitable labels are any labels detectable by an appropriate detection method. Typical labels include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels ("e.g. magnetic beads", including paramagnetic and superparamagnetic labels), and fluorescent labels.
  • Enzymatically active labels include e.g.
  • Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT- BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stock solution from Roche Diagnostics), CDP-StarTM (Amersham Biosciences), ECFTM (Amersham Biosciences).
  • a suitable enzyme-substrate combination may result in a colored reaction product, fluorescence or chemo luminescence, which can be measured according to methods known in the art (e.g. using a light-sensitive film or a suitable camera system).
  • fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes (e.g. Alexa 568). Further fluorescent labels are available e.g. from Molecular Probes (Oregon). Also the use of quantum dots as fluorescent labels is contemplated.
  • Typical radioactive labels include 35 S, 125 I, 32 P, 33 P and the like.
  • a radioactive label can be detected by any method known and appropriate, e.g. a light-sensitive film or a phosphor imager.
  • Suitable measurement methods according the present invention also include precipitation (particularly immunoprecipitation), electrochemiluminescence (electro-generated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme- linked immunosorbent assay), sandwich enzyme immune tests, electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), scintillation proximity assay (SPA), turbidimetry, nephelometry, latex- enhanced turbidimetry or nephelometry, or solid phase immune tests.
  • precipitation particularly immunoprecipitation
  • electrochemiluminescence electrochemiluminescence (electro-generated chemiluminescence)
  • RIA radioimmunoassay
  • the amount of a polypeptide may be, also preferably, determined as follows: (a) contacting a solid support comprising a ligand for the polypeptide as specified above with a sample comprising the polypeptide and (b) measuring the amount of polypeptide which is bound to the support.
  • the ligand preferably chosen from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, is preferably present on a solid support in immobilized form.
  • Materials for manufacturing solid supports include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc.
  • the ligand or agent may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
  • Suitable methods for fixing/immobilizing said ligand are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like. It is also contemplated to use "suspension arrays" as arrays according to the present invention (Nolan 2002, Trends Biotechnol. 20(l):9-12).
  • the carrier e.g. a microbead or microsphere
  • the array consists of different microbeads or microspheres, possibly labeled, carrying different ligands.
  • Methods of producing such arrays for example based on solid-phase chemistry and photo-labile protective groups, are generally known, see e.g., US 5,744,305.
  • amount encompasses the absolute amount of a polypeptide, the relative amount or concentration of the said polypeptide as well as any value or parameter which correlates thereto or can be derived therefrom.
  • values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said polypeptides by direct measurements, e.g., intensity values in mass spectra or NMR spectra.
  • values or parameters which are obtained by indirect measurements specified elsewhere in this description e.g., response levels determined from biological read out systems in response to the peptides or intensity signals obtained from specifically bound ligands. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations.
  • comparing encompasses comparing the amount of the polypeptide comprised by the sample to be analyzed with an amount of a suitable reference source specified elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount while a concentration is compared to a reference concentration or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample.
  • the comparison referred to in step (b) of the method of the present invention may be carried out manually or computer assisted.
  • the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program.
  • the computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output format. Based on the comparison of the amount determined in step a) and the reference amount, it is possible to diagnose pancreatic cancer.
  • the term "reference amounts" as used herein refers to amounts of the polypeptides which allow for determining whether a subject suffers from pancreatic cancer, or not. Therefore, the reference may either be derived from (i) a subject known to suffer from pancreatic cancer or (ii) a subject known not to suffer from pancreatic cancer, i.e. a healthy subject with respect to pancreatic cancer. Moreover, the reference amounts, preferably, define thresholds. Suitable reference amounts or threshold amounts may be determined by the method of the present invention from a reference sample to be analyzed together, i.e. simultaneously or subsequently, with the test sample. A preferred reference amount serving as a threshold may be derived from the upper limit of normal (ULN), i.e.
  • UPN upper limit of normal
  • the ULN for a given population of subjects can be determined by various well known techniques.
  • a suitable technique may be to determine the median of the population for the peptide or polypeptide amounts to be determined in the method of the present invention.
  • the reference amount in accordance with the present invention for siC3b is 6 ⁇ 1 ⁇ g/ml, more preferably, 6.1 ⁇ g/ml.
  • An amout of siC3b being larger than the reference amount is indicative for a subject suffering from pancreatic cancer.
  • siC3b is a biomarker indicative for the presence or absence of a pancreatic cancer in a subject.
  • pancreatic cancer can be determined at earlier stages wherein the pancreatic cancer elicits rather unspecific clinical symptoms. Due to the early diagnosis by the method of the present invention, therapeutic approaches can be applied earlier and may, therefore, allow for a more successful treatment of this life-threatening disease.
  • the method of the present invention allows for diagnosis with increased sensitivity and an increased positive predictive value.
  • the findings underlying the aforementioned method also allow for an improved clinical management of pancreatic cancer since patients can be identified which need intensive monitoring. Further, the success of a drug-based supportive therapy can be monitored in the adjuvant stage after resection of a pancreatic cancer.
  • 171 plasma samples from subjects treated adjuvant after resection and from healthy donors were collected prospectively and analyzed for siC3b. Patients underwent every three months imaging and the results from siC3b ELISA were categorized according to radiological-defined recurrence within six months after blood withdrawal.
  • mCRP expression on tumor cells was analyzed on cell lines after incubation with plasma from healthy donors or derived from subjects with pancreatic carcinoma.
  • C3 cleavage products were analyzed on tumor specimen by immunohistochemistry. Most important, up to six months prior to radiological defined recurrence siC3b plasma level is increased with a sensitivity of 93% which could be further increased by combining with CA 19.9. Complement regulatory proteins are high expressed on pancreatic carcinoma; CD59 expression is further increased after incubation with plasma from pancreatic carcinoma patients. Tumors express the C3b-cleavage products iC3b and C3dg. Screening for siC3b in subjects with an increased risk for pancreatic carcinoma allows early detection with high sensitivity as siC3b plasma levels are increased up to six months prior to radiological evidence. Moreover, the studies showed that sensitivity could be further increased by combination with CA 19.9. Thanks to the present invention, surgical intervention in early stage before metastatic spread has occurred or tumor mass is too extended has become possible. Moreover, in case of adjuvant treatment the therapy could be changed.
  • the present invention also relates to a method of determining whether a subject should be closely monitored for pancreatic cancer comprising the steps of a) determining the amount of siC3b in a sample of said subject; and b) comparing said amount to a reference amount, whereby a subject which should be closely monitored for pancreatic cancer is to be determined.
  • an amount being larger than the reference amount is indicative for a subject which should be closely monitored for pancreatic cancer.
  • the term "closely monitored” as used herein refers to a repeated clinical monitoring of the subject for pancreatic cancer within short periods of time.
  • said close monitoring is carried out by applying imaging-based diagnosis techniques, more preferably by CT, MRT and/or PET.
  • close monitoring is carried out within time periods of 2 weeks, one month, two months, six month or one year.
  • the method aims to determine (identify) at least a statistically significant portion of subjects which need close monitoring. However, it is not required to correctly assess any subject for need of close monitoring. Whether a portion is statistically significant can be determined by techniques recited elsewhere in this specification.
  • the present invention contemplates a method for determining whether an anticancer therapy applied to a subject after treatment of pancreatic cancer is beneficial comprising the steps: a) determining the amount of siC3b in a sample of a subject; and b) comparing said amount to a reference amount, whereby it is to be determined whether the anti-cancer therapy is beneficial for the said subject.
  • anti-cancer therapy as used herein, preferably, refers to drug based chemotherapy. More preferably, said anti-cancer therapy comprises Gemcitabine- or 5FU- based chemotherapy.
  • the aforementioned method also allows for an early determination with regard to the beneficial effects of a given therapy. Specifically, rather than by monitoring and evaluating the symptoms or clinical signs which may strongly differ in strength between the subjects and whose recording is not always possible in an objective manner, the aforementioned method of the present invention allows for a rather reliable determination of the beneficial effects of a therapy by determining the biomarker siC3b.
  • CAl 9.9 is determined.
  • CAl 9.9 refers to a carbohydrate antigen which is known to be associated with tumor malignancy, in general.
  • the CAl 9.9 antigen has been isolated and characterised as sialylated lacto-N-fucopentaose II, an oligosaccharide which is related to Lewis a blood group substance.
  • CAl 9.9 was also found to be up-regulated in patients suffering from pancreatic cancer. However, the specificity and the positive predictive value of diagnostic tests based on CA19.9, in particular for initial patient screening, is rather low.
  • a C 19.9 Assay i.e. the Roche Modular E 170 CA 19-9 electrochemiluminescent immunoassay, can be commercially purchased from Roche Diagnostics GmbH, Germany.
  • CAl 9.9 can be, preferably, determined in combination with siC3b in the methods of the present invention. Thereby, reliability and sensitivity of the methods can be even further increased.
  • threshold amounts can be determined as set forth above. Particular preferred is a threshold amount of 30 to 40 U/ml, more preferably of 38 U/ml. An amount being larger than the aforementioned threshold shall be additionally indicative for a pancreatic cancer in these preferred methods of the present invention.
  • the present invention also relates to a device for diagnosing pancreatic cancer in a subject comprising: a) means for determining the amount of siC3b in a sample of said subject; and b) means for comparing the determined amount to a reference amount, whereby pancreatic cancer will be diagnosed.
  • the term "device” as used herein relates to a system of means comprising at least the aforementioned means operatively linked to each other as to allow the diagnosis Preferred means for determining the amount of the polypeptide as well as means for carrying out the comparison are disclosed above in connection with the method of the invention. How to link the means in an operating manner will depend on the type of means included into the device. For example, where means for automatically determining the amount of the peptides are applied, the data obtained by said automatically operating means can be processed by, e.g., a computer program in order to obtain the desired results. Preferably, the means are comprised by a single device in such a case.
  • Said device may accordingly include an analyzing unit for the measurement of the amount of polypeptides in an applied sample and a computer unit for processing the resulting data for the evaluation.
  • the computer unit preferably, comprises a database including the stored reference amounts or values thereof recited elsewhere in this specification as well as a computer-implemented algorithm for carrying out a comparison of the determined amounts for the polypeptides with the stored reference amounts of the database.
  • Computer-implemented as used herein refers to a computer-readable program code tangibly included into the computer unit.
  • the means for comparison may comprise control stripes or tables allocating the determined amount to a reference amount.
  • the test stripes are, preferably, coupled to a ligand which specifically binds to the polypeptides referred to herein.
  • the strip or device preferably, comprises means for detection of the binding of said peptides or polypeptides to the said ligand.
  • Preferred means for detection are disclosed in connection with embodiments relating to the method of the invention above.
  • the means are operatively linked in that the user of the system brings together the result of the determination of the amount and the diagnostic value thereof due to the instructions and interpretations given in a manual.
  • the means may appear as separate devices in such an embodiment and are, preferably, packaged together as a kit as specified elsewhere herein in detail. The person skilled in the art will realize how to link the means without further ado.
  • Preferred devices are those which can be applied without the particular knowledge of a specialized clinician, e.g., test stripes or electronic devices which merely require loading with a sample.
  • the results may be given as output of raw data which need interpretation by the clinician.
  • the output of the device is, however, processed, i.e. evaluated, raw data the interpretation of which does not require a clinician.
  • Further preferred devices comprise the analyzing units/devices (e.g., biosensors, arrays, solid supports coupled to ligands specifically recognizing the peptide, Plasmon surface resonace devices, NMR spectrometers, mass- spectrometers etc.) and/or evaluation units/devices referred to above in accordance with the method of the invention.
  • the present invention relates to a device for determining whether a subject should be closely monitored for pancreatic cancer comprising: a) means for determining the amount of siC3b in a sample of said subject; and b) means for comparing said amount to a reference amount, whereby a subject which should be closely monitored for pancreatic cancer will be determined.
  • the present invention relates to a device for determining whether an anti- cancer therapy applied to a subject after treatment of pancreatic cancer is beneficial comprising: a) menas for determining the amount of siC3b in a sample of a subject; and b) means for comparing said amount to a reference amount, whereby it is determined whether the anti-cancer therapy is beneficial for the said subject
  • the present invention encompasses a kit for diagnosing pancreatic cancer in a subject comprising: a) means for determining the amount of siC3b in a sample of said subject; and b) means for comparing the determined amount to a reference amount, whereby pancreatic cancer will be diagnosed.
  • kit refers to a collection of the aforementioned means, preferably, provided in separately or within a single container.
  • the container also preferably, comprises instructions for carrying out the method of the present invention.
  • kits for determining whether a subject should be closely monitored for pancreatic cancer comprising: a) means for determining the amount of siC3b in a sample of said subject; and b) means for comparing said amount to a reference amount, whereby a subject which should be closely monitored for pancreatic cancer will be determined.
  • the present invention provides for a kit for determining whether an anti-cancer therapy applied to a subject after treatment of pancreatic cancer is beneficial comprising: a) means for determining the amount of siC3b in a sample of a subject; and b) means for comparing said amount to a reference amount, whereby it is determined whether the anti-cancer therapy is beneficial for the said subject.
  • Figure IAB ROC curves for siC3b, CA 19.9 and combination; CA 19.9 and siC3b were determined and correlated with the clinical status. Patients having a radiological recurrence within the next six months after blood withdrawal were declared as recurrent. Data are shown from 171 samples analyzed for siC3b, 129 samples analyzed for CA19.9 and 121 samples were both parameters were available.
  • Figure 2 ROC curves for siC3b, CA 19.9 and combination four to six prior radiological- defined recurrence; CA 19.9 and siC3b were determined and correlated with the clinical status. Only samples from patients having a radiological recurrence four to six month after blood withdrawal are included. Data are shown from 61 samples analyzed for siC3b, 45 samples analyzed for CAl 9.9 and 44 samples were both parameters were available.
  • FIG. 3 Sensitivity and specificity of siC3b and CA 19.9 in the longitudinal course; CA 19.9 and siC3b were determined and correlated with the clinical status. Data are shown from 170 samples analyzed for siC3b, 128 samples analyzed for CA19.9 and 119 samples were both parameters were available.
  • Figure 4 Time course of siC3b in 20 patients; Plasma from subjects with at least two samples from different time-points was analyzed for siC3b and evaluated according to radiological recurrence. The bold line shows mean of all 20 subjects.
  • FIG. 5 Expression of CD59 on pancreatic carcinoma cell lines after over night incubation with plasma; Three different cell lines were incubated over night either with heat-inactivated FCS or plasma derived from healthy donors or plasma withdrawn from patients suffering from pancreatic carcinoma. Cells were analyzed for expression of CD59. Data is shown as mean ⁇ standard error from at least eight separate experiments for each cell line.
  • Example 1 Sample preparation and analysis
  • Treatment protocols dealt with chemotherapy (5-FU, gemcitabine), targeted therapy (cetuximab) and chemoradio immunotherapy (CapRI scheme).
  • CT scans were performed routinely at least every three months, or in case of clinical symptoms. The interval between blood withdrawal and radiological evidence of recurrence was calculated.
  • ASPC-I, Pane 1 and Capan-1 (all PDAC cell lines) were purchased from DSMZ (Deutsche Sammlung fur Mikroorganismen und Zellkultur, Braunschweig, Germany).
  • the cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS, PAA, C ⁇ lbe, Germany), 100 U/ml penicillin and lOO ⁇ g/ml streptomycin (Seromed, J ⁇ lich, Germany).
  • paraffin-embedded tissue sections (3-5 ⁇ m thick) were subjected to immunostaining. Tissue sections were deparaffinized in Roticlear (Carl Roth GmbH, Düsseldorf, Germany) and rehydrated in progressively decreasing concentrations of ethanol. Slides were placed in washing buffer (10 nM Tris-HCl, 0.85% NaCl, 0.1% BSA, pH 7.4) and subjected to immunostaining.
  • tissue section After antigen was retrieved by boiling the tissue section in 10 mM citrate buffer for 10 min in the microwave oven, the sections were incubated first with 3% peroxidase (in methanol) for 10 min and then washed and incubated with 3% BSA (in TBS) for 1 hr to block non-specific binding sites.
  • Tissues were incubated with mouse monoclonal anti iC3b covered, human polyclonal C3d antibody covers diluted 1 :800 in washing buffer, or with corresponding IgG as control at 4°C overnight.
  • the slides were rinsed with washing buffer and incubated with HRP-labeled anti-rabbit antibodies (DAKO Cytomation, Hamburg, Germany) for 45 min at room temperature.
  • the slides were washed in washing buffer and each section was subjected to 100 ⁇ l DAB- chromogen substrate mixture (DAKO), and then counterstained with Mayer's haematoxylin.
  • the sections were washed, dehydrated in progressively increasing concentrations of ethanol, and mounted with xylene-based mounting medium. Staining in the cancer cells of each sample was categorized as absent, weak, moderate or strong. Mann- Whitney test was used on SPSS 11.5 was used to analyze statistical significance where appropriate. A/?-value ⁇ 0.05 was considered as significant.
  • Example 2 Positive predictive value, sensitivity and specificity of siC3b as a biomarker
  • Example 3 Modulation of mCRPs on pancreatic carcinoma cells by patient-derived plasma
  • CD46, CD55 and CD59 were determined.
  • the cell lines expressed highly CD46 (median 100%, IQR 78%; 100%) and CD55 (median 79%; IQR 41%; 89%) under standard culture conditions with heat-inactivated FCS. This was not affected by incubation with non heat- inactivated (i.e.

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Abstract

La présente invention concerne le domaine du diagnostic de cancer. En particulier, la présente invention concerne un procédé permettant le diagnostic du cancer du pancréas chez un sujet comprenant les étapes de détermination de la quantité de siC3b dans un échantillon d’un sujet et la comparaison de ladite quantité à une quantité de référence, permettant ainsi le diagnostic du cancer du pancréas. La présente invention concerne également un procédé permettant de déterminer si un sujet doit être suivi de près pour un cancer du pancréas et un procédé permettant de déterminer si une thérapie anticancéreuse appliquée au sujet après le traitement du cancer du pancréas est bénéfique. L’invention concerne en outre des dispositifs et des trousses de diagnostic pour la mise en œuvre desdits procédés.
PCT/EP2009/056951 2008-06-16 2009-06-05 Procédés et moyens de diagnostic du cancer du pancréas WO2009153175A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016030426A1 (fr) * 2014-08-26 2016-03-03 Roland Andersson Procédé de détermination probabiliste de prédisposition d'un patient au cancer du pancréas

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WO2007102526A1 (fr) * 2006-03-02 2007-09-13 Oncotherapy Science, Inc. Procédés de diagnostic du cancer du pancréas utilisant la protéine reg4

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WO2007102526A1 (fr) * 2006-03-02 2007-09-13 Oncotherapy Science, Inc. Procédés de diagnostic du cancer du pancréas utilisant la protéine reg4

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JAN SCHMIDT ET AL: "Release of iC3b from apoptotic tumor cells induces tolerance by binding to immature dendritic cells in vitro and in vivo", CANCER IMMUNOLOGY, IMMUNOTHERAPY, SPRINGER, BERLIN, DE, vol. 55, no. 1, 27 October 2005 (2005-10-27), pages 31 - 38, XP019422473, ISSN: 1432-0851 *
LIN YIING ET AL: "Proteins associated with disease and clinical course in pancreas cancer: a proteomic analysis of plasma in surgical patients.", JOURNAL OF PROTEOME RESEARCH SEP 2006, vol. 5, no. 9, September 2006 (2006-09-01), pages 2169 - 2176, XP002538845, ISSN: 1535-3893 *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016030426A1 (fr) * 2014-08-26 2016-03-03 Roland Andersson Procédé de détermination probabiliste de prédisposition d'un patient au cancer du pancréas
CN107003371A (zh) * 2014-08-26 2017-08-01 瑞可安诊断公司 用于确定主体患胰腺癌的可能性的方法

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