WO2002037113A2 - Validation clinique et fonctionnelle de cibles a partir de cellules cancereuses disseminees - Google Patents

Validation clinique et fonctionnelle de cibles a partir de cellules cancereuses disseminees Download PDF

Info

Publication number
WO2002037113A2
WO2002037113A2 PCT/EP2001/012786 EP0112786W WO0237113A2 WO 2002037113 A2 WO2002037113 A2 WO 2002037113A2 EP 0112786 W EP0112786 W EP 0112786W WO 0237113 A2 WO0237113 A2 WO 0237113A2
Authority
WO
WIPO (PCT)
Prior art keywords
cancer cells
target
status
individuals
disseminated
Prior art date
Application number
PCT/EP2001/012786
Other languages
English (en)
Other versions
WO2002037113A3 (fr
Inventor
Michael Giesing
Hansjörg GRILL
Beatrix Boeckmann
Bernhard Suchy
Original Assignee
Michael Giesing
Grill Hansjoerg
Beatrix Boeckmann
Bernhard Suchy
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from DE2000154635 external-priority patent/DE10054635A1/de
Application filed by Michael Giesing, Grill Hansjoerg, Beatrix Boeckmann, Bernhard Suchy filed Critical Michael Giesing
Priority to AU2002216009A priority Critical patent/AU2002216009A1/en
Publication of WO2002037113A2 publication Critical patent/WO2002037113A2/fr
Publication of WO2002037113A3 publication Critical patent/WO2002037113A3/fr

Links

Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells

Definitions

  • the present invention relates to methods for the cancer-related clinical validation of a target from disseminated cancer cells, to methods for the functional validation of said targets, to methods for treating cancer taking account of the status of at least one of said validated targets and to kits for target-related cancer treatment. Building on this, target-related development of active substances is possible.
  • Cancer cells enter the bloodstream in particular during the course of vascularization of a solid tumor. They circulate in the bloodstream and likewise in other body fluids and may form metastases according to their molecular endowment.
  • cytokeratin (CK) 20 mRNA expression in blood indicate that the detection of disseminated cancer cells by a CK20-specific RT PCR assay is related to the stage of disease (Fujii Y et al., Jpn J Cancer Res 1999 Jul; 90 (7): 753-757; Denis MG et al., Int J Cancer 1997 Oct 21 ; 74 (5): 540-544; Soeth E et al., Cancer Res 1997 Aug 1 ; 57 (15): 3106-3110; Soeth E et al., Int J Cancer 1996 Aug 22; 69 (4): 278-282; Gudemann C J et al., J Urology 2000 Aug; 164(2): 532-536) and that detection of epithelial cells in preoperative blood was associated with reduced disease-free and overall survival (Hardingham JE et al., Int J Cancer 2000 Jan 20; 89 (1) :8-13).
  • CK20-mRNA was an useful marker to detect circulating cancer cells in breast cancer but there was no correlation between CK-20 mRNA expression and stage or auxiliary lymph node status (Bae JW et al, J Korean Med Sci 2000 Apr; 15 (2) : 194-198) or there was no correlation between the detection of CK20 mRNA in the peripheral blood and disease progression and survival in patients with known metastatic colorectal cancer (Wyld DK et al., Int J Cancer 1998 Jun 19; 79 (3): 288-292).
  • PSA prostate-specific antigen
  • ⁇ -fetoprotein fails to correlate with disease stage (Kienle P et al., Arch Surg 2000 Feb; 135 (2) : 213-218; Hautkappe AL et al., Cancer Res 2000 Jun 15; 60(12): 3170-3174; Barbu V et al., Hepatology 1997 Nov; 26(5): 1171-1175) but seems to correlate with recurrence in patients with hepatocellular carcinoma (Wong IH et al., Clin Cancer Res 1999 Dec; 5 (12) 4021- 4027).
  • HER2/neu mRNA is reported to correlate with the stage of breast cancer (Wasserman L et al., Mol Diagn 1999 Mar; 4 (1) : 21-28).
  • CEA mRNA in the blood is said to be a useful indicator of circulating tumor cells, and to be one of the determinant prognostic factors for patients with colorectal carcinoma (Taniguchi et al., Cancer 2000 Sep 1 ; 89 (5) : 970-976) , and to correlate with a significant shorter survival in patients with non-small cell lung cancer (Yamashita Jl et al., J Thorac Cardiovasc Surg 2000 May; 119(5): 899-905).
  • the simulataneous presence of CEA and CK20 mRNA is said to be a potent prognostic factor (Yamaguchi K et al., Ann Surg 2000 Jul; 232(1 ): 58-65).
  • WO 99/10528 describes a method for identifying and characterizing disseminated cancer cells with the help of multiple-parameter analyses of cancer-specific and cancer-associated genes.
  • the relevance of such genes to cancer generally arises from a multiplicity of studies which connect clinically diagnosed cancers with one of said genes.
  • LOH loss of heterocygosity
  • disseminated cancer cells for example the identification of novel therapeutic targets; in the context of developing active substances, the screening of active substances, for example identifying and characterizing lead substances and in particular for developing active substances against surface structures of disseminated cancer cells; the choice of a therapy depending on the particular individual and on the stage of the disease.
  • This object is achieved according to the invention by the assessment of targets from disseminated cancer cells with respect to their role for the clinical manifestation of cancers.
  • the present invention therefore relates to a method for the clinical validation of a target from disseminated cancer cells, which method comprises that for a population of individuals it is determined whether a target status determined in disseminated cancer cells of the individuals correlates with at least one cancer-related information about the clinical status of the individuals.
  • target denotes any biochemical or molecular biological feature of disseminated cancer cells. This includes both therapeutic and diagnostic targets and in particular prognostic targets. Genomic targets at DNA level, for example, are included as well as targets involved in expression, for example those at RNA level, in particular at mRNA level or at protein level. Examples of targets may be mutations, insertions, deletions, LOHs, amplifications, aberrations in the chromosomal set, and the like; expression of splice variants; and also over- and underexpression of particular mRNAs or proteins - and other unusual, in particular cancer-specific modifications of particular cell components.
  • Preferred targets are those which affect qualitative features of the DNA machinery and/or RNA machinery. Special mention must be made here of targets which affect and, in particular, oncogenically influence cell properties such as cell division, cell growth, cell-cell interactions, inhibition of tumor suppression and therapy resistances and thus have an influence on the clinical picture of a cancer. Targets relating to DNA recombination, DNA amplification, DNA repair, cell cycle inducers and apoptosis inhibitors are in particular included here.
  • the targets to be validated according to the invention may be targets known per se. Examples which may be mentioned are:
  • oncogenes and tumor suppressor genes such as p53, genes of the ras family erb-B2, c-myc, md ⁇ m2, c-fos, DPC4, FAP, nm23, RET, WT1, and the like, LOHs, for example with regard to p53, DCC, APC, Rb and the like and also BRCA1 and BRCA2 in hereditary tumors, microsatellite instability of MSH2, MLH1 , WT1 and the like; also tumorous RNAs such as CEA, cytokeratins, e.g.
  • CK20, BCL-2, MUC1 in particular tumor-specific splice variants hereof, MAGE3, Mud 8, tyrosinase, PSA, PSM, BA46, Mage-1 and the like, or else morphogenic RNAs such as maspin, hCG, GIP, motilin, hTG, SCCA-1 , AR, ER, PR, various hormones and the like;
  • RNAs and proteins which affect the etastasizing profile i.e. the expression of molecules involved in angiogenesis, motility, adhesion and matrix degradation such as bFGF, bFGF-R, VEGF, VEGF-Rs, such as VEGF-R1 or VEGF-R2, E-cadherin, integrins, selectins, MMPs, TIMPs, SF, SF-R and the like, the cell cycle profile or proliferation profile, such as cyclins (e.g.
  • cyclins D, E and B expression ratio of cyclins D, E and B), Ki67, p120, p21 , PCNA and the like, or the apoptosis profile, such as FAS (L+R), TNF (L+R), perforin, granzyme B, BAX, bcl-2, caspase 3 and the like.
  • a particular aspect of the present invention relates to the identification of targets from disseminated cancer cells and to the validation thereof.
  • targets may have a relevance with respect to cancer which is known and which is related to, for example, primary tumor material or which is not established.
  • identification is aimed, for example, at DNA aberrations such as mutations, amplifications and LOHs.
  • splice variants are identified in disseminated cancer cells.
  • Splice variants are expression products of particular genes whose mRNA is alternatively spliced. Accordingly, splice variants differ qualitatively, for example with regard to composition and/or sequence of exons and/or by additional incorporation of intron (partial) sequences. According to the previously mentioned modifications, splice variants may occur at mRNA level and in particular may lead to a shift in the reading frame or to the generation of a stop codon. Likewise, they may occur at the protein level and may lead to expression of a protein with modified structure and function, in particular to expression of a truncated protein.
  • splice variants are identified by using at least one exon-spanning primer for amplifying a particular mRNA or a transcript thereof, in particular cDNA.
  • two or more exon-spanning primers may be used.
  • the amplicons from disseminated cancer cells are compared with amplicons obtained accordingly from normal cells. Normal cells generally mean cells without cancerous modifications. For studying disseminated cancer cells from blood, the preferred normal cells used are leukocytes. The comparison makes it possible to identify amplicons and thus also mRNAs of normal and altered size.
  • the composition of the amplicons and thus of the mRNAs may be determined in a manner known per se, for example by hybridization, in particular on biochips with or without prior amplification, or by sequencing.
  • This embodiment for identifying splice variants is directed especially at genes having cancer-related function, i.e. in particular genes affecting qualitative features of the DNA machinery and/or RNA machinery.
  • cancer-related function i.e. in particular genes affecting qualitative features of the DNA machinery and/or RNA machinery.
  • cell-cycle-related genes such as cyclins B, D and E, p21, p53 or the apoptosis-related genes such as caspases 1 to 9, bax, bcl-2, Fas ligand or Fas receptor.
  • splice variants are identified by amplifying mRNA or a transcript thereof, in particular cDNA, by means of degenerate PCR primers specific for conserved, functional domains of particular protein families, for example src domain, SH2 domain or kinase domain. Comparison with accordingly amplified mRNA or a transcript thereof, in particular cDNA, of normal cells leads, according to the previously described procedure when using exon-spanning primers, to the identification of splice variants affecting conserved, functional domains of proteins.
  • splice variants are identified by means of differential hybridization known per se, preferably subtraction hybridization (SSH).
  • SSH subtraction hybridization
  • cDNA from disseminated cancer cells and cDNA from normal cells may be compared and hybridized against a cDNA gene library.
  • the gene library may have been generated either from the disseminated cancer cells or from the normal cells. Preference is given to hybridizing tester cDNA from disseminated cancer cells against driver cDNA from normal cells. It is in principle also possible to use poly(A) RNA or sense cRNA instead of the driver cDNA.
  • the hybridization generally uses an excess of cDNA and/or poly(A) RNA or sense cRNA of one of the two cell populations over the other. It is preferred to use an excess of the appropriate nucleic acids from normal cells, i.e. in particular of driver cDNA.
  • the tester cDNA is initially cut by a restriction enzyme prior to hybridization.
  • the tester cDNA is ligated in two mixtures with two different adapters which represent the binding sites for the primers of a later PCR.
  • Each mixture of the adapter-carrying tester cDNA is hybridized with an excess of driver cDNA.
  • the non-hybridizing and therefore still single-stranded DNAs or RNAs of interest may be identified and, if required, isolated, and in particular sequenced.
  • sequences left which are present in the tester cDNA, but not in the driver cDNA.
  • These transcripts can be amplified in a subsequent PCR. To this end, the two adapter mixtures are combined.
  • This embodiment especially relates to the identification of splice variants of genes to which no cancer-related function has been assigned yet.
  • the clinical validation of the invention is based on the assignment of target and clinic.
  • a particular biochemical or molecular biological feature and on the other hand characteristics of a cancer, in particular , as clinical symptoms.
  • the present invention also relates to a method for collecting a cancer- related dataset (profile) of an individual, characterized in that a) the individual is examined for disseminated cancer cells, b) optionally at least one aliquot of a sample derived from the body of the individual examined in a) is set aside in a substance library, c) at least one cancer-related information about the clinical status of the individual is provided, and d) the result obtained from the examination in a) and the information provided in c) and, optionally, the sample set aside in b) are linked to each other in the form of a dataset.
  • the examination of an individual for disseminated cancer cells includes an investigation of whether or not a sample from the body of the individual comprises disseminated cancer cells.
  • this examination of disseminated cancer cells also comprises determination of the status of at least one target from the disseminated cancer cells. For the purpose of clinically validating a target, the status of this target is expediently determined.
  • the "status of a target” comprises any appearance of a particular target with respect to quantity and/or quality. Whether determining the status provides a qualitative or quantitative result depends especially on the definition of the target. Qualitative determination of the status is generally directed toward the investigation of whether or not a particular target is present. Determining the status of these targets may be carried out, for example, in such a way that the result is either "positive” or “negative”. To this end, it is necessary to define the target in such a way that a particular set of biochemically and/or molecular biologically determinable analytical values is assigned to the results "positive” or "negative”.
  • qualitative determination of the status generally also includes quantitative determination of values, which the definition of the target is based on. It is also possible, for example, to define the overexpression or underexpression of particular mRNAs or proteins as a target in such a way that expressed amounts of a particular mRNA or protein above or below a limit are to be judged "positive” or “negative”. According to the invention, qualitative determination of the status is useful, since it simplifies the determination, which is based on statistical evaluation, of whether the target status correlates with the cancer-related information.
  • examples may be the detection of particular DNA sequences, in particular of mutated sequences (positive/negative); amounts of particular sequences compared with wild-type sequences or reference sequences or when compared to the amount of the same sequence in non-cancer cells, preferably derived from the same individual and in particular from the same sample, in particular in case of amplifications (e.g. positive, if ratio > 2) and LOHs (e.g.
  • mRNA sequences positive/negative
  • splice variants positive/negative
  • the presence of particular mRNAs in disseminated cancer cells positive/negative
  • amounts of particular mRNAs in particular reduced or enhanced expressions of mRNAs (positive above a particular amount of mRNA per cell equivalent or when compared to the amount of the same mRNA in non-cancer cells, preferably derived from the same individual and in particular from the same sample); or the presence or absence of a particular protein (positive/negative).
  • cancer cell means according to the invention a cell having one or more modifications connected with cancer, i.e. degeneration in a general sense. This definition is based on the idea that generation of cancer is a continuous modifying process.
  • the term cancer cell therefore also includes precursors of cancer cells and in particular tumor cells, which have cancerous or tumorous modifications.
  • disseminated cancer cell is defined in particular in relation to solid tumors, i.e. especially primary tumors, metastases and recurrences.
  • disseminated cancer cells can circulate in the body of an individual. This generally involves transport organs of the body, especially body fluids and in particular blood.
  • Disseminated cancer cells generally derive from a solid tumor by being initially part of a solid tumor, i.e. in particular of tumor tissue, from which they subsequently detach.
  • disseminated cancer cells are characterized by their relatively low amount with respect to non-cancer cells equally present. They are therefore also denoted as residual cancer cells (minimal residual disease, or MRD for short).
  • residual cancer cells minimal residual disease, or MRD for short.
  • the proportion of disseminated cancer cells is generally below 1 :1000, usually below 1 :10,000 and in many cases even below 1:100,000, with respect to the number of non-cancer cells in a randomly obtained sample of body fluid. For blood, these ratios are valid in particular with respect to mononuclear cells (MNC for short).
  • disseminated cancer cells are studied in cell-containing mixtures having disseminated cancer cells optionally in addition to non-cancer cells.
  • the mixtures may have different proportions of disseminated cancer cells.
  • cancer cell contents of 50% or more may be useful. Accordingly it is preferred to use preparations derived from a body fluid of the individual which have a cancer cell to non-cancer cell ratio of more than 1 :1. Higher ratios of more than 9:1 and in particlar cases of more than 99:1 are even more preferred for determining the status of the target and to assign said status to the cancer cells.
  • the enrichment of disseminated cancer cells can be carried out in a manner known per se, for example via immunospecific adsorption methods or, preferably, using size- and/or shape-dependent separation techniques such as the screening technique described in WO 00/06702 which is incorporated herein by reference.
  • the latter method using a screen that retains said cancer cells has the advantage that the disseminated cancer cells are recovered in an essentially unaltered state which reflects the biological state they have in their natural environment, i.e. the body fluid of the individual they have been derived from.
  • WO 99/40221 and WO 00/46585 also incorporated herein by reference describe methods for the enrichment of tumor cells by means of density gradient centrifugation using separation mediums having a defined density in the range of 1 ,055 to 1 ,070 g/ml or 1 ,055 to 1 ,065 g/ml, respectively.
  • the status of a particular target can be determined in a manner known per se. Choosing suitable methods is within the skilled worker's knowledge and is based in many cases on extraction measures, on amplification reactions of particular nucleic acids, in particular by means of polymerase chain reaction (PCR for short), where appropriate combined with reverse transcription (RT for short), on hybridization experiments, for example from the well-established Southern blot to the use of biochips, and also on immunological detection reactions.
  • PCR polymerase chain reaction
  • RT reverse transcription
  • This aliquot may be a part of the sample taken from the body of the individual, in particular of a body fluid such as blood.
  • the substance library comprises for a sample taken from an individual at a particular point in time at least two preparations: a first which has been enriched for disseminated cancer cells and a second which is substantially free of disseminated cancer cells.
  • tissue samples for example mediastinoscopy, bone marrow biopsy, removal of axillary lymph nodes, laparotomy
  • blood values for example with respect to blood levels of particular enzymes such as alkaline phosphatase, lactate dehydrogenase or acidic phosphatase, or of the sugar level
  • visualizing examinations in particular CT and MRI, and ultrasound examinations.
  • the tumor classification represents usually particularly important cancer-related information about the clinical status of an individual. Mention must be made here especially of TNM classification which is known per se and classifies tumors according to their anatomic extent.
  • TNM classification which is known per se and classifies tumors according to their anatomic extent.
  • One aspect of this system relates to the assessment of this primary tumor itself. Of particular importance according to the invention are the statements “no indication of a primary tumor (TO)” and “size of the primary tumor (T1-T4)”.
  • Another aspect relates to the assessment of regional lymph nodes. Of particular importance here are the statements “no regional lymph node metastases (NO)” and “metastatic involvement of regional lymph nodes (N1-N3)”.
  • Another aspect relates to the assessment of distant metastases, the statements “no distant metastases (MO)” and “distant metastases, stating the attacked organ(s), where appropriate (M1)” being of particular importance here.
  • Yet another aspect relates to the assessment of residual tumors (local recurrences).
  • the statements “no residual tumour (RO)”, “microscopic residual tumour (R1 )” and “macroscopic residual tumor (R2)” are particularly important according to the invention.
  • Another aspect of staging relates to the assessment of the histopathological degree of differentiation (grading), the statements “well differentiated (G1)”, “moderately differentiated (G2)”, “badly differentiated (G3)” to “undifferentiated (G4)” being particularly important according to the invention.
  • a type of cancer-related information which is preferred according to the invention is determined by the moment when the individual dies. This moment may be related to a first point in time, for example to the moment when a cancer, for example a primary tumor, a local recurrence or a distant metastasis, is diagnosed, to the moment when disseminated cancer cells are taken, in particular when the target status is determined, or when a therapeutic treatment commences or ends.
  • the information is generally given in form of a period which is to be denoted according to the invention by the term "survival time".
  • the period relates to the first, in principle deliberately selectable point in time, preferably the moment of making the diagnosis or, in particular, of taking disseminated cancer cells from the individual. In the case of a plurality of individuals, the information is given as an average.
  • survival time it is also possible to give the survival rate, i.e. the percentage of individuals surviving a particular period.
  • cancer-free i.e. tumor-free and in particular recurrence- free
  • this period is determined by a first point in time, for example the time of making the diagnosis, the moment when a cancer of an individual is, according to the criteria above, not diagnosable (any longer), for example after removing the tumor, and/or the moment of taking disseminated cancer cells.
  • this period is determined by a second point in time when in the same individual a cancer, i.e. in particular a renewed tumor (recurrence), where appropriate as local recurrence or as distant metastasis, is or is not diagnosed.
  • the first point in time is in principle deliberately selectable under the above conditions. It is expediently determined by the moment of removing the tumor or, in particular, of the taking of disseminated cancer cells.
  • the second point in time more or less defines the clinical endpoint and indicates the clinical importance of cancer-related information. Examples of suitable statements are "no tumor”, “tumor after less than 2 years” and “tumor after 2-5 years”.
  • a particular item of cancer-related information is the qualitative and, optionally appropriate, also quantitative assessment of disseminated cancer cells of an individual.
  • the status of one or more than one target (geno- and phenotyp) of the disseminated cancer cells which, once clinically validated according to the invention, reflects the item(s) of cancer-related information it correlates with.
  • a comparison in this regard at various times and, based thereupon, the finding of whether the amount and/or type of disseminated cancer cells have changed, in particular increased or decreased, generally with respect to one or more parameters, in particular targets. This information especially reflects the progress of the cancer and can provide direct evidence for taget-related therapeutic effectiveness.
  • a further particular aspect of the present invention relates to the indication of therapeutic measures as cancer-related information.
  • suitable information are "no therapy”, in particular "no surgery”, “no systemic therapy”; “therapy”, in particular “surgery” or “systemic therapy”, e.g. "chemotherapy, optionally indicating the active substance(s) and/or therapy plan”, “radiation (radiotherapy)", “adjuvant therapy”, for example “hormone therapy”, “immunomodulation” or “target-related therapy”, or combinations thereof.
  • steps c) and d) are repeated for the same individual at another time, i.e. the individual is again clinically examined and the cancer-related information obtained is added to the already existing dataset (follow-up).
  • steps a) and optionally b) and also d) may be repeated for the same individual at another time (longitudinal assessment) so that a plurality of samples from an individual, which have been taken and optionally set aside at different times, can be added to the dataset.
  • the substance library includes samples which have disseminated cancer cells or cell components derived therefrom and which were taken from the same individual at various times. It is then advantageously possible to assign cancer-related information about the clinical status of an individual to samples, which have been taken at different times, for example based on the moment of taking the samples. Moreover, it is advantageous to provide the possibility to facilitate a time-based comparison.
  • the target status which is determinable in disseminated cancer cells of an individual is assigned to at least one item of cancer- related information about the clinical status of the individual, where appropriate with respect to the moment when the disseminated cancer cells were taken. This assignment is carried out for a plurality of individuals.
  • the database may be defined to comprise datasets which assign one or more than one item of clinical information to one or more than one target status.
  • Databases in accordance with the invention are collections or compilations of data, numbers, facts or other elements which may be compiled, stored and accessed using electronic, electromagnetic, electrooptical or similar methods. Said elements are arranged systematically or methodically and are individually accessible (structure). The database also includes those elements which are necessary for running or using the database, for example thesaurus or indexing systems.
  • the present invention therefore also relates to such databases, the building and use thereof for clinical validation of targets from disseminated cancer cells.
  • the data comprise at least one item of cancer-related information for each of various individuals and the assignment of this information to disseminated cancer cells of each individual.
  • the data also comprise the status of at least one target from the disseminated cancer cells.
  • it comprises the status of more than one target from disseminated cancer cells.
  • the database makes it possible to group individuals on the basis of one or more item of cancer-related information and, where appropriate, also on the basis of the status of one or more targets from the disseminated cancer cells. Setting up the database includes adding cancer-related information about individuals already recorded in the database and also adding further individuals.
  • Adding information of further individuals leads to the corresponding addition of disseminated cancer cells from said individuals or, where appropriate, of information about the status of one or more targets from the disseminated cancer cells. If further cancer-related information about individuals already recorded in the database is added, then it may be useful to add further disseminated cancer cells or, where appropriate, information about the status of one or more targets from said disseminated cancer cells.
  • a population is thus a group of individuals having at least one cancer-related common feature.
  • Such a group is formed, for example, by individuals having a particular history such as mastophathy or a diagnosed and possibly surgically removed tumor of a particular type, for example patients having had breast carcinoma surgery.
  • the status of the target from corresponding disseminated cancer cells is assigned to each of the individuals of this population. This assignment makes it possible to deduce the proportion of individuals of this population, who have a particular target status, compared with the total number of individuals in this population.
  • this target status correlates with at least one further item of cancer-related information about the clinical status of the individuals is based on a comparison.
  • a subpopulation is formed among the individuals included in the population, using said further cancer-related information about the clinical status.
  • the status of the target from corresponding disseminated cancer cells is again assigned to this subpopulation. This assignment makes it possible to deduce the proportion of individuals of a specific population who have the target status. Comparison of the proportion of individuals of the population firstly with the proportion of individuals of the subpopulation and secondly with respect to a particular target status forms the basis for the correlation analysis.
  • the individuals may be divided up into the following groups, depending on the time of determination of the staus: patients with precancerous lesions; patients having a manifest tumor (which may have been treated, e.g. surgically removed, or not) prior to systemic therapy; patients after systemic therapy, in particular with a short-term observation of from about 3 to 24, preferably 5 to 12 and in particular 6 to 10 months, a medium-term observation of from 2 to 5 years or a long-term observation of more than 5 years after systemic therapy.
  • Individuals with recurrence can be included as a further group which in turn can be furher devided up into individuals before systemic therapy and individuals after systemic therapy.
  • these groups of individuals may then be divided up using further cancer-related information, for example the survival time and preferably the cancer-free period. Accordingly it can be determined whether or not survival time or cancer-free period correlates with the status of a target in one of these groups. This may also provide information on the choice of therapy if, for instance, survival time or cancer-free period correlates with status of the target for a population of individuals having undergone a particular therapy while there is no correlation in other populations of individuals having undergone a different therapy or no therapy.
  • further cancer-related information for example the survival time and preferably the cancer-free period. Accordingly it can be determined whether or not survival time or cancer-free period correlates with the status of a target in one of these groups. This may also provide information on the choice of therapy if, for instance, survival time or cancer-free period correlates with status of the target for a population of individuals having undergone a particular therapy while there is no correlation in other populations of individuals having undergone a different therapy or no therapy.
  • the invention relates to a method for clinically validating the therapeutic relevance of a target from disseminated cancer cells, which comprises determining whether a status of said target correlates with at least one item of cancer-related information about the clinical status of at least one population of individuals who have undergone a first systemic therapy and at least for one further population of individuals who have undergone no or a different systemic therapy.
  • Said item of cancer-related information preferably is selected from survival time, cancer-free period or changes of the amount of disseminated cancer cells having said target status.
  • the above groups of individuals are divided up into at least 3 subgroups having a particular target status: (a) no tumor (primary or recurrence, respectively) within the observation period (b) early tumor (primary or recurrence, respectively), e.g. within the first half of the observation period, in particular after less than 2 years from the target status determination (c) late tumor (primary or recurrence, respectively), e.g. within the second half of the observation period, in particular after 2-5 years.
  • the observation period advantageously starts when the individuals are examined for disseminated cancer cells, i.e. the status of the target to be evaluated is determined. Moreover, it is also possible to select other points in time, e.g. the time of diagnosis of the tumor or its removal by surgery.
  • Disseminated cancer cells characterized by such a molecular algorithm can be directly used to validate further targets from disseminated cancer cells. Since said algorithms reflect at least one item of cancer-related information about the clinical status of the corrsponding individual, a correlation between the status of a particular target and one of said algorithms indicates a correlation between the status of said target and said cancer-related information.
  • the evaluation by correlation analysis is carried out using statistics, in particular by means of suitable bio-mathematical methods, for example by logistic regression (cf. J.
  • the target is clinically validated in the case of a statistical significance of p ⁇ 0.10 with 90% significance, preferably of p ⁇ 0.05 with 95% significance and in particular of p ⁇ 0.01 with 99% significance.
  • This clinical validation relates in a narrower sense to the connection between the presence in an individual of disseminated cancer cells characterized according to the target status, and the individual's clinical status represented by the cancer-related information included in the evaluation.
  • this also means a connection between the target as a feature of disseminated cancer cells and the clinical picture of a cancer.
  • the clinical validation according to the invention of a target includes in particular the finding of whether the evaluated target status leads to progression of the cancer (driver) or protects against it (protector). This finding is of principal importance for diagnosis and prognosis, and especially for a target-specific cancer therapy.
  • driver targets and protector targets may be made, for example, using survival time analysis.
  • the survival function describing the survival time indicates the probability of surviving until a particular point in time.
  • the statistical estimation of the survival function may be carried out, for example, using the Kaplan- Meier method (also called product limit).
  • the survival functions firstly of individuals having a particular status of the target to be evaluated and secondly of individuals not corresponding to this status are compared using the log rank test or the Wilcoxon test.
  • the clinical validation of the invention In suitable cases and in view of a treatment carried out on an individual, it is possible for the clinical validation of the invention to facilitate distinguishing between those individuals who respond to the treatment (responders) and those who do not (non- responders). This finding too is of fundamental importance for the choice of therapy and in the end ensures increased effectiveness on a particular therapeutic measure, e.g. on administration of particular active substances.
  • the purpose of the method of the invention is the clinical validation of targets from disseminated cancer cells. If the determination according to the method results in a correlation between the target status and cancer-related information, then the target is clinically validated according to the invention. Depending on the correlating cancer- related information, the clinical validation comprises a connection of particular aspects of the clinical picture of cancers with the target status.
  • the dependence on defined clinical endpoints such as the death of the individual and/or the appearance of recurrences is determined (validation as an prognostic indicator), where appropriate taking into account particular therapeutic measures, in particular a systemic therapy (validation as a therapeutic indicator).
  • the present invention aims at the clinical validation of targets from disseminated cancer cells as independent indicators, i.e. where the target status does not correlate with primary tumor-related information, such as the TNM-claasification.
  • combinations of a plurality of targets are clinically validated.
  • the finding that the status of a first target in combination with the status of one or more further targets correlates with at least one item of cancer- related information about the clinical status of an individual is carried out in analogy to the above-described procedure.
  • a particular combination of statuses with respect to a plurality of targets is assigned to the populations or subpopulations. This results in molecular algorithms which can be used to validate further targets, even without having recourse to the individuals clinic status, simply be determining whether the status of the target to be evaluated correlates with a particular molecular algorithm.
  • the clinical validation is carried out by comparison with controls which have no disseminated cancer cells carrying the target to be validated.
  • Targets from disseminated cancer cells which have been clinically validated according to the invention, then form the basis for target-related dealing with cancers. This includes an appropriate diagnosis and a target-related therapy.
  • the present invention further relates to a method for the functional validation of the targets, preferably of targets clinically validated according to the invention.
  • the active substance is allowed to act ex vivo on disseminated cancer cells whose reaction is then determined. It is, for example, possible to choose at least one measurement parameter whose modification is determined in the disseminated cancer cells.
  • the disseminated cancer cells expediently correspond to a particular target status; in particular they possess this target (positive).
  • the measures of the method may be carried out accordingly on cells not corresponding to the target status, i.e.
  • the above method also allows testing active substances on targets from disseminated cancer cells and functionally validating them. This applies in particular to target-related active substances. This is an important aspect of active substance development according to which potential active substances can be selected in a target-related manner - for example in the context of screening methods - and subsequently be validated.
  • the purpose of this method for the functional validation of target and/or active substance is detecting active-substance-dependent, molecular and/or morphological modifications in the disseminated cancer cells. If the determination of one or more parameters results in a state in the disseminated cancer cells which, after the action of the active substance, is different from the state prior to the action of the active substance, then the target is functionally validated with respect to the active substance, or the active substance is functionally validated with respect to the target, according to the invention.
  • the functional validation comprises in particular a functional connection between active substance and target in disseminated cancer cells.
  • targets may be cloned and expressed in a manner known per se.
  • suitable cell systems in particular human cell lines, which can be transfected or transformed appropriately, are available to the skilled worker.
  • target-carrying cell systems may be contacted with one or more active substances in the manner already described above. This method too serves to establish a molecular and/or morphological algorithm of action, which in turn can be validated according to the invention in disseminated cancer cells.
  • active substances having antisense function, antibody function or ribozyme function are preferred.
  • This type of active substances is advantageous according to the invention, since they can be specifically developed and optimized with respect to a target clinically validated according to the invention.
  • active substances having antisense function, antibody function or ribozyme function may also serve to characterize biological control systems, in particular biological regulatory circuits. This in turn may lead to the identification of a drug target which is different from the actual target, which has been clinically and functionally validated with respect to the antisense molecule, immunoglobulin or ribozyme used, but interacts with the latter via the characteristic control system.
  • progesterone receptor expression in disseminated cancer cells is clinically validated according to the invention.
  • Estrogen receptor expression is dependent on progesterone receptor expression. Both are regulated via a shared control system.
  • the estrogen receptor is a suitable drug target which may be treated, for example, with tamoxifen.
  • the biological control system may be characterized in established tumor cell lines, normal cells or, preferably, in expediently isolated disseminated tumor cells. Methods for characterizing a biological control system are known per se, for example knockout or knockdown of a parameter using antisense molecules, biochemical analysis, determination of gene expression using well-established methods or biochips, and bioinformatics. Characterization of the biological control system may identify upstream or downstream drug targets which, owing to their structure (e.g. enzymes, receptors), serve as drug targets for developing, for example, specific enzyme inhibitors or receptor blockers.
  • targets on the cancer cell surface may be accessible to both antisense approaches and antibody approaches, while antisense molecules in particular are available for intracellular targets.
  • Useful antisense molecules are characterized by their specific binding to the target RNA. Expediently, at least parts of the antisense sequence are complementary to partial sequences of the RNA. Preference is given to using nucleic acids which are not cleaved by particular cellular enzymes, in particular RNases. Furthermore, a certain rigidity is advantageous. Thus LNAs (locked nucleic acids) are particularly preferred.
  • Useful active substances having antibody function are in particular immunoglobulins, for example antibodies or functional fragments thereof, in particular human, animal, polyclonal, monoclonal and recombinant immunoglobulins, antibody fragments, for example Fab, Fab', F(ab) 2 , synthetic immunoglobulins. They are in particular characterized by binding to protein targets. This binding advantageously takes place in such a way that the cancer-related relevance of the target is reduced.
  • a typical example is Herceptin.
  • the clinical and functional validation of a target from disseminated cancer cells provides an advantageous basis for developing and testing target-related active substances.
  • the focus on targets clinically and functionally validated uniformly in disseminated cancer cells allows active substance development including pharmacogenomic and toxicogenomic aspects to reduce unwanted side effects and including a correct stratification of patients, i.e. an, if necessary time-dependent, individualized application of active substances.
  • active substance development including pharmacogenomic and toxicogenomic aspects to reduce unwanted side effects and including a correct stratification of patients, i.e. an, if necessary time-dependent, individualized application of active substances.
  • substantial savings in costs and time are obtained.
  • the targets clinically and also, where appropriate, functionally validated according to the invention offer the advantage to have the possibility of an individualized therapy.
  • the present invention also relates to a method for cancer treatment, where the status of at least one validated target from disseminated cancer cells of an individual is determined and a therapy is chosen depending on the status.
  • the determination of the status represents a diagnostic measure and relates in particular to the characterization of disseminated cancer cells from body fluids, in particular blood.
  • the characterization includes both the identification and detection of the cancer cells as such and the determination of one or more parameters in these cancer cells.
  • this procedure relates in particular to the method for characterizing disseminated and micrometastasized cancer cells using DNA and/or RNA as described in WO 99/10528.
  • Part of the treatment method of the invention is therefore also a method for characterizing disseminated cancer cells using DNA, RNA and/or proteins, in which the cancer cells which, if required, have been enriched from the body fluid of an individual are tested for at least one clinically and preferably also functionally validated target. It is advantageous and in particular cases useful to carry out the same test on non-cancer cells of the same individual for comparison. This method does not exclude further diagnostic measures.
  • the choice of therapy falls into the expanded field of diagnosis, too. It allows choosing from particular therapies an optimal therapy plan which includes the type of active substance(s), dosage and administration schedule, while weighing up the benefit/risk ratio.
  • the choice of therapy thus includes in particular the choice between radiotherapy, chemotherapy and adjuvant therapy, the choice of the active substance class or of particular active substances, for example antiandrogens, antiestrogens, aromatase inhibitors, Herceptin, Panorex, antifolates, 5-FU/FA, 6-thiopurines, taxanes, cisplatin and analogs, anthracyclines, metalloprotease inhibitors, angiogenesis inhibitors, differentiation-inducing active substances, nonspecific immunomodulators, and the like, and the choice of target-related active substances.
  • the choice of therapy includes a choice of dosage depending on the disseminated cancer cells, in particular depending on the amount and/or the status of disseminated cancer cells of the individual to be treated.
  • a particular aspect of this method relates to the possibility of therapy monitoring according to which the status of at least one validated target is repeatedly determined at different times and the therapy already selected is assessed and, if necessary, adjusted depending on the state and in particular depending on changes in the status.
  • a method of this type expediently includes the status of a plurality of targets, too.
  • the treatment per se i.e. carrying out one or more therapeutic measures, is directed according to the invention at disseminated cancer cells and the indications linked thereto and is generally based on the use of at least one therapeutic agent, in particular one active substance in an effective amount for administration, or on the use of this therapeutic agent, in particular active substance, for preparing an appropriate agent.
  • Appropriate agents usually in the form of pharmaceutical compositions comprising said active substance and optionally further acitve substances and/or pharmaceutically ingredients for suitable formulation , such as carriers, diluents etc., are well known in the art.
  • kits for target-related cancer therapy which comprise - agents for determining the status of at least one validated target in disseminated cancer cells of an individual; - instructions for the therapy taking account of the determined status.
  • the user may have recourse to the appropriate therapeutic agent, generally an active-substance-containing pharmaceutical.
  • kits of the invention relate to the combination of firstly a diagnostic component related to disseminated cancer cells and secondly a therapeutic component.
  • disseminated cancer cells are a tumor entity independent of the primary tumor or metastases and form, according to the invention, the basis for a more sophisticated and more efficient cancer therapy.
  • a particular advantage is gained from clinically validating a target even before clinical testing in the conventional sense, in particular before phase II.
  • Another advantage is the possibility of being able to test active substances on humans ex vivo, and this is of great importance, in particular for identifying lead substances and especially for the validation thereof prior to the complicated preclinical phase of pharmacology and toxicology.
  • a considerably shorter time for for developing active substances for more effective treatment of cancer is ensured than is currently the case.
  • the present invention relates to a method for clinically validating the expression of cytokeratin 20 (target).
  • This validation relates especially to carcinomas.
  • the determination of the status of this target is based on the positive or negative detection of cytokeratin-20 mRNA in cell-containing body fluids, in particular blood, or in fractions derived therefrom, in particular the MNC fraction, for example by means of RT-PCR.
  • the individuals are generally patients having carcinomas which have been diagnosed and, where appropriate, already surgically treated, in particular patients having breast carcinomas. Information about the primary tumor, therapy measures and recurrence status of a plurality of individuals is provided.
  • cytokeratin-20 mRNA correlates with the early appearence of recurrences.
  • negative cytokeratin-20-mRNA expression correlates with chemotherapeutic and radiotherapeutic measures, in particular with anthracycline (AC) therapy.
  • AC anthracycline
  • the result of clinically validating cytokeratin-20 mRNA from disseminated cancer cells is that cytokeratin 20-expressing disseminated tumor cells are accessible to a combination of chemotherapy and radiotherapy.
  • chemotherapy according to the AC plan has a cytoreductive effect.
  • a corresponding method for clinically validating CK19 mRNA results, with respect to the same individuals, in no significant correlation between CK19 mRNA expression of disseminated cancer cells and chemotherapy, radiotherapy or hormone therapy or combinations thereof.
  • cytokeratin-20 mRNA opens up the possibility of providing target-related active substances, in particular antisense molecules complementary to cytokeratin-20 mRNA; of testing active substances on cytokeratin 20 mRNA-expressing disseminated cancer cells and determining ex vivo the effect of a particular active substance on cytokeratin 20 mRNA expression; moreover, of treating carcinomas, in particular gynecological carcinomas such as breast carcinomas and ovarian carcinomas by determining cytokeratin-20 expression in disseminated cancer cells of an individual and, in the case of a positive result, choosing a combination of chemotherapy and radiotherapy, with chemotherapy preferably carried out according to the AC plan.
  • target-related active substances in particular antisense molecules complementary to cytokeratin-20 mRNA
  • the present invention relates to a method for clinically validating expression of the progesterone receptor (target).
  • This validation relates especially to carcinomas.
  • the determination of the status of this target is based on the positive or negative detection of PR mRNA in cell-containing body fluids, in particular blood, or in fractions derived therefrom, in particular the MNC fraction, for example by means of RT-PCR.
  • the individuals are generally patients having carcinomas which have been diagnosed and, where appropriate, already surgically treated, in particular patients having breast carcinomas. Information about the primary tumor, therapy measures and recurrence status of a plurality of individuals is provided.
  • the result of clinically validating PR mRNA from disseminated cancer cells is that PR-expressing disseminated tumor cells are accessible to a hormone therapy, in particular a therapy using tamoxifen. This provides the possibility of identifying those patients who respond to tamoxifen (responders).
  • the present clinical validation of PR mRNA opens up the possibility of providing target-related active substances, in particular antisense molecules complementary to PR mRNA; of testing active substances on PR mRNA-expressing disseminated cancer cells and determining ex vivo the effect of a particular active substance on PR mRNA expression; moreover, of treating carcinomas, in particular gynecological carcinomas such as breast carcinomas and ovarian carcinomas by determining PR expression in disseminated cancer cells of an individual and, in the case of a positive result, choosing a hormone therapy.
  • a diagnostic method comprises dertermining PR mRNA expression in disseminated cancer cells of an individual wherein a positive result indicates a good prognosis, in particular a low risk for recurrences and a prolonged survival time.
  • the present invention relates to a method for clinically validating overexpression of bcl-2 (target).
  • This validation relates especially to carcinomas.
  • the determination of the status of this target is based on the detection of bcl-2 mRNA.
  • the status is positive if, in a cell fraction that is enriched for disseminated cancer cells obtained from cell-containing body fluids, in particular blood, or from fractions derived therefrom, in particular the MNC fraction, more bcl-2 mRNA is detected - for example by means of RT-PCR - than in a cell fraction tested for comparison which has a relatively low cancer cell content and thus is, in particular, substantially free of disseminated cancer cells.
  • the individuals are generally patients having carcinomas which have been diagnosed and, where appropriate, already surgically treated, in particular patients having breast carcinomas.
  • Information about the primary tumor, therapy measures and recurrence status of a plurality of individuals is provided.
  • the evaluation of a sufficiently large population of individuals shows no significant correlation with conventional, histological classification parameters, such as lymph node status, grading or tumor size.
  • overexpression of bcl-2 mRNA correlates with an increased number of recurrences and with chemotherapy and hormone therapy, in particular tamoxifen therapy.
  • the probability of recurrences is substantially higher for detection of increased bcl-2- mRNA expression than for negative detection or in comparison with the detection of normal expression in benign control cells from the patients.
  • bcl-2 mRNA opens up the possibility of providing target-related active substances, in particular antisense molecules complementary to bcl-2 mRNA; of testing active substances on bcl-2 mRNA-overexpressing disseminated cancer cells and determining ex vivo the effect of a particular active substance on bcl-2 mRNA expression; moreover, of treating carcinomas by determining bcl-2 expression in disseminated cancer cells of an individual and, in the case of a positive result, choosing radiotherapy rather than chemotherapy or hormone therapy, in particular rather than tamoxifen therapy.
  • a diagnostic method comprises dertermining bcl-2 overexpression in disseminated cancer cells of an individual wherein a positive result indicates a poor prognosis, in particular a high risk for recurrences and a reduced survival time.
  • the present invention relates to a method for clinically validating expression of ErbB2, the combined expression of CyclinB and -D, the combined expression of ErbB2, CyclinB and -D, and the combined expression of ErbB2, CyclinB, -D and bcl-2 (targets).
  • This validation relates especially to carcinomas.
  • the status is positive if, in a cell fraction that is enriched for disseminated cancer cells obtained from cell-containing body fluids, in particular blood, or from fractions derived therefrom, in particular the MNC fraction, more mRNA is detected - for example by means of RT-PCR - than in a cell fraction tested for comparison which has a relatively low cancer cell content and thus is, in particular, substantially free of disseminated cancer cells.
  • the individuals are generally patients having carcinomas which have been diagnosed and, where appropriate, already surgically treated, in particular patients having breast carcinomas. Information about the primary tumor, therapy measures and recurrence status of a plurality of individuals is provided.
  • the result of clinically validating ErbB2, CyclinB, -D and/or bcl-2 mRNA from disseminated cancer cells, carried out according to the invention is that ErbB2- and bcl-2-overexpressing disseminated tumor cells are accessible to antibody therapy.
  • adjuvant therapy with herceptin has a cytoreductive effect.
  • the inventive clinical validation of ErbB2, CyclinB, -D and/or bcl-2 mRNA opens up the possibility of providing further target-related active substances, in particular antisense molecules complementary to said mRNAs; of testing active substances on ErbB2, CyclinB, -D and/or bcl-2 mRNA-expressing disseminated cancer cells and determining ex vivo the effect of a particular active substance on ErbB2, CyclinB, -D and/or bcl-2 mRNA expression; moreover, of treating carcinomas, in particular gynecological carcinomas such as breast carcinomas and ovarian carcinomas by determining ErbB2, CyclinB, -D and/or bcl-2 overexpression in disseminated cancer cells of an individual and, in the case of a positive result, choosing an adjuvant therapy, preferably with a target-related active substance such as Herceptin.
  • a target-related active substance such as Herceptin.
  • a diagnostic method comprises dertermining CyclinB, -D and/or bcl-2 overexpression in disseminated cancer cells of an individual wherein a positive result indicates a poor prognosis, in particular a high risk for recurrences and a reduced survival time.
  • Figure 1 shows the dependence of the proportion of those patients without recurrence on the recurrence-free period in months for patients having positive PR mRNA detection ( ) and patients having negative PR mRNA detection
  • Figure 2 shows the dependence of the proportion of those patients without recurrence on the recurrence-free period in months for patients having bcl-2 mRNA overexpression ( ) and patients having normal bcl-2 mRNA expression ( — );
  • Figure 3 shows the dependence of the proportion of those patients without recurrence on the recurrence-free period in months for patients having ErbB2 mRNA overexpression ( ) and patients having normal ErbB2 mRNA expression (-
  • Figure 4 shows the dependence of the proportion of those patients without recurrence on the recurrence-free period in months for patients having CyclinB and CyclinD mRNA overexpression ( ) and patients having normal
  • Figure 5 shows the dependence of the proportion of those patients without recurrence on the recurrence-free period in months for patients having combined overexpression of ErbB2, CyclinB and CyclinD mRNA ( ) and patients having normal ErbB2 and CyclinB and CyclinD mRNA expression ( );
  • Figure 6 shows the dependence of the proportion of those patients without recurrence on the recurrence-free period in months for patients having combined overexpression of ErbB2, bcl-2, CyclinB and/or CyclinD mRNA ( ) and patients having normal expression of these parameters ( );
  • MNC Mononuclear cells
  • RNA is isolated from the MNC fraction in a manner known per se and transcribed into cDNA by means of random hexamers and reverse transcriptase.
  • CK20 mRNA-specific detection is then carried out by means of PCR and using the 5'-nuclease detection system known per se (TaqMan®). The following primer sequences and probe sequences were used: SEQ ID NO:1-3.
  • As a control blood from healthy individuals was analyzed accordingly.
  • MNC Mononuclear cells
  • RNA is isolated from the MNC fraction in a manner known per se and transcribed into cDNA by means of random hexamers and reverse transcriptase. PR-specific detection is then carried out by means of PCR and using the 5'-nuclease detection system known per se (TaqMan®). The following primer sequences and probe sequences were used: SEQ ID NO:7-9.
  • MNC Mononuclear cells
  • B CD 45-positive isolates from the sieve flow-through
  • C sieve residue, cancer cell fraction having a disseminated cancer cell content of at least 1 :1 are obtained from the MNC fraction.
  • RNA is then isolated from fractions A and C in a manner known per se and transcribed into cDNA by means of random hexamers and reverse transcriptase.
  • Bcl-2-specific detection is then carried out by means of PCR and using the 5'-nuclease detection system known per se (TaqMan®). The following primer sequences and probe sequences were used: SEQ ID NO:4-6.
  • SEQ ID NO:4-6 SEQ ID NO:4-6.
  • the ratio of bcl mRNA cell equivalents to GAPDH cell equivalents is formed for each of fractions A and C, and the ratio of the resulting quotients is in turn formed. If the ratio of fraction-C quotient to fraction-A quotient is greater than 1, bcl-2 mRNA is overexpressed.
  • the cell equivalents are based on a cell standard. This cell standard is prepared by extracting RNA from a known number of cells (e.g.
  • RNA 2 x 10 "s ) from a cell suspension of a carcinoma cell line expressing the particular parameter, and transcribing said RNA into cDNA.
  • This cDNA is present in each quantitative analysis in the form of a dilution series (e.g. 6 dilution levels) and serves as a reference system.
  • MNC Mononuclear cells
  • B CD 45-positive isolates from the sieve flow-through
  • C sieve residue, cancer cell fraction having a disseminated cancer cell content of at least 1 :1 are obtained from the MNC fraction.
  • RNA is then isolated from fractions A and C in a manner known per se and transcribed into cDNA by means of random hexamers and reverse transcriptase.
  • bcl-2-specif ⁇ c detetcion was carried out as described in Example 3.
  • ErbB2-, CyclinB- and CyclinD-specific detection is then carried out by means of PCR and using the 5 ' -nuclease detection system known per se (TaqMan®). The following primer sequences and probe sequences were used: SEQ ID NO:10-18.
  • SEQ ID NO:10-18 SEQ ID NO:10-18.
  • RNA from a known number of cells (e.g. 2 x 10 s ) from a cell suspension of a carcinoma cell line expressing the particular parameter, and transcribing the said RNA into cDNA.
  • This cDNA is present in each quantitative analysis in the form of a dilution series (e.g. 6 dilution levels) and serves as a reference system.
  • lymph node status There is no correlation of lymph node status, grading, tumor size or menopausal status with ErbB2-, CyclinB- and CyclinD-mRNA overexpression.
  • Herceptin therapy of >1 year correlates with reduced detection of both ErbB2 and bcl- 2 mRNA.
  • Table 2 Comparison of target status and of parameters for the histological tumor classification according to the TNM system and of the menopausal status in all patients
  • Table 3 Comparison of target status and of parameters for the histological tumor classification according to the TNM system and of the menopausal status in patients recurrence-free for at least 2 years
  • Table 5 Comparison of target status and therapeutic treatments in patients recurrence-free for at least 2 years
  • Table 7 Comparison of bcl-2 mRNA overexpression and appearance of recurrences for various therapeutic treatments
  • Table 8 Overexpression of CyclinD, CyclinB and bcl-2 mRNA in patients with ErbB2 overexpression.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Hospice & Palliative Care (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de validation clinique, en relation avec le cancer, d'une cible à partir de cellules cancéreuses disséminées, des procédés de validation fonctionnelle des cibles, des méthodes de traitement du cancer prenant en compte l'état d'au moins une des cibles validées, ainsi que des nécessaires destinés au traitement de cancer se rattachant à une cible.
PCT/EP2001/012786 2000-11-03 2001-11-05 Validation clinique et fonctionnelle de cibles a partir de cellules cancereuses disseminees WO2002037113A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002216009A AU2002216009A1 (en) 2000-11-03 2001-11-05 Clinical and functional validation of targets from disseminated cancer cells

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US24585400P 2000-11-03 2000-11-03
DE10054635.8 2000-11-03
DE2000154635 DE10054635A1 (de) 2000-11-03 2000-11-03 Klinische und funktionelle Validierung von Targets disseminierter Krebszellen
US60/245,854 2000-11-03

Publications (2)

Publication Number Publication Date
WO2002037113A2 true WO2002037113A2 (fr) 2002-05-10
WO2002037113A3 WO2002037113A3 (fr) 2004-02-19

Family

ID=26007559

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/012786 WO2002037113A2 (fr) 2000-11-03 2001-11-05 Validation clinique et fonctionnelle de cibles a partir de cellules cancereuses disseminees

Country Status (2)

Country Link
AU (1) AU2002216009A1 (fr)
WO (1) WO2002037113A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1803822A1 (fr) * 2005-12-30 2007-07-04 Adnagen AG Procédé pour la caractérisation individuelle de molécules cibles thérapeutiques, et leur utilisation
WO2009094619A1 (fr) 2008-01-25 2009-07-30 Cytotech Labs, Llc Système de tests pour évaluer l'oncogénicité, l'évolution d'une tumeur et l'efficacité d'un traitement
EP2287329A3 (fr) * 2004-07-09 2011-08-03 University of Pittsburgh of the Commonwealth System of Higher Education Identification de marqueurs du cancer de l'ýsophage
EP3199641A1 (fr) * 2016-01-27 2017-08-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédés permettant d'évaluer l'évolution, de saisir les données et de traiter une maladie cancéreuse
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DENIS M G ET AL: "Detection of disseminated tumor cells in peripheral blood of colorectal cancer patients." INTERNATIONAL JOURNAL OF CANCER. JOURNAL INTERNATIONAL DU CANCER. UNITED STATES 21 OCT 1997, vol. 74, no. 5, 21 October 1997 (1997-10-21), pages 540-544, XP002207545 ISSN: 0020-7136 *
GIESING M ET AL: "Independent prognostication and therapy monitoring of breast cancer patients by DNA/RNA typing of minimal residual cancer cells." THE INTERNATIONAL JOURNAL OF BIOLOGICAL MARKERS. ITALY 2000 JAN-MAR, vol. 15, no. 1, January 2000 (2000-01), pages 94-99, XP002207543 ISSN: 0393-6155 *
IINUMA H ET AL: "Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and K-ras genes in patients with colorectal cancer." INTERNATIONAL JOURNAL OF CANCER. JOURNAL INTERNATIONAL DU CANCER. UNITED STATES 20 JUL 2000, vol. 89, no. 4, 20 July 2000 (2000-07-20), pages 337-344, XP002207544 ISSN: 0020-7136 *
PELKEY J P ET AL: "Molecular and immunological detection of circulating tumor cells and micrometastases from solid tumors" CLINICAL CHEMISTRY, AMERICAN ASSOCIATION FOR CLINICAL CHEMISTRY. WINSTON, US, vol. 42, no. 9, 1996, pages 1369-1381, XP002092613 ISSN: 0009-9147 *
VAN DER VELDE-ZIMMERMANN D ET AL: "Molecular test for the detection of tumor cells in blood and sentinel nodes of melanoma patients" AMERICAN JOURNAL OF PATHOLOGY, PHILADELPHIA, PA, US, vol. 149, no. 3, 1 September 1996 (1996-09-01), pages 759-764, XP002092617 ISSN: 0002-9440 *
WYLD D K ET AL: "Detection of colorectal cancer cells in peripheral blood by reverse-transcriptase polymerase chain reaction for cytokeratin 20." INTERNATIONAL JOURNAL OF CANCER. JOURNAL INTERNATIONAL DU CANCER. UNITED STATES 19 JUN 1998, vol. 79, no. 3, 19 June 1998 (1998-06-19), pages 288-293, XP002207546 ISSN: 0020-7136 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2287329A3 (fr) * 2004-07-09 2011-08-03 University of Pittsburgh of the Commonwealth System of Higher Education Identification de marqueurs du cancer de l'ýsophage
WO2007076989A1 (fr) * 2005-12-30 2007-07-12 Adnagen Ag Méthode de caractérisation individuelle de molécules cibles thérapeutiques et applications
EP1803822A1 (fr) * 2005-12-30 2007-07-04 Adnagen AG Procédé pour la caractérisation individuelle de molécules cibles thérapeutiques, et leur utilisation
JP2015052605A (ja) * 2008-01-25 2015-03-19 バーグ リミテッド ライアビリティ カンパニー 腫瘍原性、腫瘍進行、および処置効率の査定のためのアッセイシステム
EP2245460A4 (fr) * 2008-01-25 2011-05-25 Cytotech Labs Llc Système de tests pour évaluer l'oncogénicité, l'évolution d'une tumeur et l'efficacité d'un traitement
EP2245460A1 (fr) * 2008-01-25 2010-11-03 Cytotech Labs, LLC Système de tests pour évaluer l'oncogénicité, l'évolution d'une tumeur et l'efficacité d'un traitement
EP2708894A2 (fr) * 2008-01-25 2014-03-19 Berg LLC Système de dosage pour l'évaluation de l'oncogénicité, progression des tumeurs et efficacité du traitement
EP2708894A3 (fr) * 2008-01-25 2014-04-02 Berg LLC Système de dosage pour l'évaluation de l'oncogénicité, progression des tumeurs et efficacité du traitement
WO2009094619A1 (fr) 2008-01-25 2009-07-30 Cytotech Labs, Llc Système de tests pour évaluer l'oncogénicité, l'évolution d'une tumeur et l'efficacité d'un traitement
US10365279B2 (en) 2008-01-25 2019-07-30 Berg Llc Assay system for the assessment of oncogenicity, tumor progression, and treatment efficacy
US10260104B2 (en) 2010-07-27 2019-04-16 Genomic Health, Inc. Method for using gene expression to determine prognosis of prostate cancer
CN108770360A (zh) * 2016-01-27 2018-11-06 弗劳恩霍夫应用研究促进协会 对癌性疾病进行分期、分型和治疗的手段和方法
JP2019508032A (ja) * 2016-01-27 2019-03-28 フラウンホーファーゲゼルシャフト ツール フォルデルング デル アンゲヴァンテン フォルシユング エー．フアー． がん性疾患をステージング、タイピングおよび処置するための手段および方法
WO2017129753A1 (fr) 2016-01-27 2017-08-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédés de stadification, typage et traitement d'une maladie cancéreuse
EP3199641A1 (fr) * 2016-01-27 2017-08-02 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Moyens et procédés permettant d'évaluer l'évolution, de saisir les données et de traiter une maladie cancéreuse
CN108770360B (zh) * 2016-01-27 2022-06-14 弗劳恩霍夫应用研究促进协会 对癌性疾病进行分期、分型和治疗的手段和方法
US11702701B2 (en) 2016-01-27 2023-07-18 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Means and methods for staging, typing and treating a cancerous disease

Also Published As

Publication number Publication date
WO2002037113A3 (fr) 2004-02-19
AU2002216009A1 (en) 2002-05-15

Similar Documents

Publication Publication Date Title
Bozionellou et al. Trastuzumab administration can effectively target chemotherapy-resistant cytokeratin-19 messenger RNA–positive tumor cells in the peripheral blood and bone marrow of patients with breast cancer
MeJean et al. Prognostic factors of renal cell carcinoma
Lacroix Significance, detection and markers of disseminated breast cancer cells
Bianchini et al. Comparative study of gene expression by cDNA microarray in human colorectal cancer tissues and normal mucosa
JP5971769B2 (ja) アントラサイクリン療法を用いて乳癌を処置する方法
Min et al. S100A4 expression is associated with lymph node metastasis in papillary microcarcinoma of the thyroid
Yuan et al. Expression of the folate receptor genes FOLR1 and FOLR3 differentiates ovarian carcinoma from breast carcinoma and malignant mesothelioma in serous effusions
JP6144695B2 (ja) タキサン療法を用いて乳癌を処置する方法
Vaziri et al. Breast tumor immunophenotype of BRCA1-mutation carriers is influenced by age at diagnosis
WO2004032842A2 (fr) Sous classification moleculaire de tumeurs renales et decouverte de nouveaux marqueurs diagnostiques
Chami et al. BRAF mutations in pediatric metanephric tumors
US20130259858A1 (en) Signature for Predicting Clinical Outcome in Human HER2+ Breast Cancer
Dai et al. Role of miR-139 as a surrogate marker for tumor aggression in breast cancer
Fang et al. Identification of differentially expressed genes in human breast cancer cells induced by 4-hydroxyltamoxifen and elucidation of their pathophysiological relevance and mechanisms
CN109715830A (zh) 用于使用基因表达谱诊断肺癌的组合物和方法
WO2010119126A2 (fr) Outils et méthodes de diagnostic permettant de prédire l'efficacité d'agents ciblés contre l'activation de la voie de l'igf-1 dans le cancer
Alshareeda et al. Cancer of unknown primary site: real entity or misdiagnosed disease?
EP4110955A1 (fr) Procédés de diagnostic, de pronostic et de gestion du traitement du cancer du sein
Huper et al. Isogenic normal basal and luminal mammary epithelial isolated by a novel method show a differential response to ionizing radiation
Babeto et al. Differentially expressed genes in giant cell tumor of bone
Sa-Ngiamwibool et al. Usefulness of NF2 hemizygous loss detected by fluorescence in situ hybridization in diagnosing pleural mesothelioma in tissue and cytology material: A multi-institutional study
US20140100188A1 (en) Phenotyping tumor-infiltrating leukocytes
JP2012515538A (ja) ２つの遺伝子の発現の観察による乳癌患者の予後診断
WO2002037113A2 (fr) Validation clinique et fonctionnelle de cibles a partir de cellules cancereuses disseminees
Blanes et al. Kinetic profiles by topographic compartments in muscle-invasive transitional cell carcinomas of the bladder: role of TP53 and NF1 genes

Legal Events

Date Code Title Description
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP