WO2000070352A1 - Traitement du cancer - Google Patents

Traitement du cancer Download PDF

Info

Publication number
WO2000070352A1
WO2000070352A1 PCT/GB2000/001866 GB0001866W WO0070352A1 WO 2000070352 A1 WO2000070352 A1 WO 2000070352A1 GB 0001866 W GB0001866 W GB 0001866W WO 0070352 A1 WO0070352 A1 WO 0070352A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
p9ka
protein
cells
agent
Prior art date
Application number
PCT/GB2000/001866
Other languages
English (en)
Inventor
Philip Spencer Rudland
Barry Roger Barraclough
Hilmar Meek Warenius
Original Assignee
The University Of Liverpool
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 GBGB9911302.9A external-priority patent/GB9911302D0/en
Priority claimed from GB0007254A external-priority patent/GB0007254D0/en
Application filed by The University Of Liverpool filed Critical The University Of Liverpool
Priority to AU45981/00A priority Critical patent/AU4598100A/en
Publication of WO2000070352A1 publication Critical patent/WO2000070352A1/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
    • 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

Definitions

  • the present invention concerns methods and assays for determining a more accurate prognosis for patients suffering from cancer.
  • the present invention also relates to methods and assays for selecting the most appropriate therapy for patients suffering from cancer.
  • the invention is particularly concerned with determining a prognosis for patients having a cancer with cells that express p9Ka.
  • the invention also particularly relates to methods for selecting an agent for treating cancer, which agent is more effective in patients expressing p9Ka than patients not expressing p9Ka.
  • breast cancer The most common cancer found in women in Europe, the United States of America, Australia and many Latin-American countries is breast cancer. In Europe breast cancer constitutes 20 % of all cancers in women amounting to 180,000 cases per year. In the USA there are 130,000 cases per year. Since the incidence of breast cancer has increased in most countries at a rate of 1-2 % per year, it has been predicted that the annual incidence of breast cancer will have risen to 1,000,000 new cases by early in the next millennium. Other common cancers show similar increases in incidence. Despite recent advances, breast and other commonly occurring cancers still carry a high mortality. For example in breast cancer 30-40 % of new cases will be expected to die as a result of this disease.
  • the breast cancer cells break off from the primary tumour and metastasise via the lymphatic and blood vessels.
  • the initial spread of cancer cells is usually to the local lymph nodes, most frequently to the adjacent axilla. Thereafter breast cancer cells can disseminate via the blood stream resulting in distant metastases, which are usually the cause of death in the majority of breast cancer patients.
  • the presence of metastatic cancer cells in the lymph nodes has been the most widely used indicator of a poor prognosis for the patient (Harris et al 1994).
  • patients who do not have clinical evidence of lymphatic spread of the disease from the primary cancer develop metastatic disease at distant sites in the body, suggesting that this test is not entirely reliable.
  • Chemotherapy has been used in the treatment of breast cancer for over two decades.
  • chemotherapy consists of giving the patient combinations of cytotoxic drugs which have been selected on the basis of their effectiveness as single agents.
  • cytotoxic drugs One of the most effective single agents against breast cancer is doxorubicin, an anthracycline cytotoxic drug derived from the micro-organism Streptomyces peucetus var caesius.
  • Doxorubicin belongs to a broad class of cytotoxic drugs termed intercalating agents.
  • Tumours are generally understood to be detectable clinically only when their constituent cells have divided sufficiently to bring their total number to about 10 ⁇ . Similarly metastases do not become clinically apparent until a similar number of cells have accumulated as a result of uncontrolled cell division. At this stage chemotherapy can cause remissions but cannot be given in sufficiently high a dose to cure the disease without compromising growing populations of normal cells to the extent that death would ensue. The policy has thus arisen to give chemotherapy to patients who are likely to have distant metastases when the few cells that may have spread to distant sites in the body have not had sufficient time to grow to large numbers. This type of chemotherapy is termed adjuvant chemotherapy.
  • protems that are up-regulated are the products of the so-called dominant oncogenes (Sinn et al., 1987), whilst others, which are down-regulated, are the products of tumour suppressor genes (Lee et al., 1996).
  • tumour suppressor genes are concerned with the regulation of cell growth, examples, of which are Ras, Myc, EGF, ErbB, ErbB2 and p53. Whilst these proteins may be important in the initial development of a tumour, and may contribute to the evolution of further genetic changes to the cancer cells, they are probably more associated with the early stages of development of a tumour and are not associated solely with the later metastatic spread of cancer cells.
  • proteins that are associated with the fidelity of replication and DNA repair (Lengauer et al, 1998). Such proteins allow further damage to the genetic material of the cancer cells to accumulate because their mutated counterparts fail to repair damage to the genetic material of a cell. Such changes tend to have an important effect early in the development of a tumour, permitting further alterations in other proteins critical for its development.
  • This disparate group comprises previously characterised proteins as well as others of unknown normal function.
  • this group consists of enzymes such as proteases (Durfy, 1992), proteins associated with cell adhesion (Iwamura et al, 1997) and motility factors (Cajot et al, 1997; Meyer- Siegler and Hudson, 1996).
  • osteopontin An example of a protein thought to be associated with adhesion is osteopontin (Oates et al, 1996, 1997; Chen et al., 1997) and one associated with motility is a regulatory calcium-ion-binding protein, p9Ka (S100A4) (Barraclough et al, 1982; Barraclough et al., 1984; Barraclough et al, 1987; Ebralidze et al, 1989; Gibbs et al., 1994).
  • p9Ka As an example of a metastasis-inducing protein, p9Ka has been shown to induce metastatic capability in non-metastatic rat and mouse mammary tumour cells.
  • p9Ka also termed S100A4 and h-mtsl
  • osteopontin are genes whose over-expression can cause metastasis in experimental rodent models of breast cancer.
  • Over-expression of p9Ka has also been suggested to be associated with metastatic spread to local lymph nodes in human breast cancer (Albertazzi et al., 1998), and shown to be present in liver metastases arising from colon carcinomas (Takanaga et al, 1997).
  • nm23 A gene has also been described which can suppress metastatic potential. Some metastatic cells are understood to express low levels of nm23. The potential relationship of nm23 to chemotherapeutic response has been studied by Freigy et al (1997). In human breast cancer and melanoma cell lines, they examined the cytotoxic and cytostatic effects of 171 compounds in clinical use or under development. No significant relationship between nm23 expression and responsiveness to any of the 171 agents was found. Thus, at the present time there is no indication of whether expression of specific genes related to metastasis (e.g. p9Ka and osteopontin) can influence chemotherapeutic response to the extent that one anti-cancer agent is predictably more effective in treating cancer than another anti-cancer agent.
  • specific genes related to metastasis e.g. p9Ka and osteopontin
  • this invention provides a clinical assay for determining a prognosis for a subject having a cancer, which assay comprises:
  • step (b) comparing the level or abundance determined in step (a) with a pre-determined correlation between p9Ka level or p9Ka protein abundance and survival of subjects having cancer;
  • the subject in the present case is not especially limited, and may be an animal such as a mammal. Most preferably, however, the subject is a human subject. Similarly, the type of cancer is not especially limited, but it is preferred that the cancer is a human cancer.
  • the sample is obtained by extracting it from the subject.
  • This extraction may, for example, take the form of a biopsy, or may alternatively be a blood sample.
  • the sample need not comprise blood cells.
  • the sample may be processed by performing PCR, RTPCR or Northern blotting for determining p9Ka.
  • a correlation in the context of the present invention is a relationship between subject (patient) survival and the level of p9Ka or p9Ka protein in the cancer cells.
  • Patient survival may take the form of average survival time of patients, or percentage of patients surviving at a certain time, or any other form that reflects survival in a study group of cancer patients.
  • the correlation is a correlation between p9Ka level or p9Ka protein abundance and length of survival time of patients having cancer. More preferably, the correlation is a correlation determined by excluding patients from the study group that die from causes other than their cancer.
  • the correlation is a correlation between p9Ka level or p9Ka protein abundance and survival of subjects having the same cancer as the subject being assayed.
  • the correlation need not be limited in this way, and may be a correlation between p9Ka levels and survival of patients having, e.g., a metastatic cancer generally.
  • Some extremely useful and predictive correlations have been provided by the present inventors using clonogenic assays, and further correlations can be provided, if desired, using this method.
  • Types of correlation are not especially limited, provided that the data obtained for the correlation is statistically predictive.
  • correlations may relate to survival of specific types of subject, limited (for example) by age or sex, and correlations may relate to specific classes of cancer, such as metastatic cancers and/or solid tumours, or to specific cancers, such as breast cancer.
  • the present correlations indicate, in a statistically predictive manner, that the higher the p9Ka level, the worse the prognosis for the patient.
  • the correlations which are direct correlations between p9Ka levels and survival, have been found by the inventors to be many times more statistically significant, and hence more predictive, than indicators studied previously, such as metastatic spread of cancer cells to axillary nodes (see e.g. Albertazzi et al., 1998)
  • Figure 1 shows that p9Ka over-expression detected immunocytochemically by a polyclonal antibody (+) in specimens of primary breast cancer in a cohort of patients carries a very poor prognosis in comparison with a cohort of patients with specimens with unstained cells ( ⁇ 1%) (-) (graph A) even when only weakly detectable at borderline levels (1-5%) (+/-) (graph B);
  • Figure 2 shows the results of clonogenic assays, in which over-expression of p9Ka in transfected rat mammary (Rama) 37 cells is accompanied by increased sensitivity to doxorubicin;
  • Figure 3 shows correlations between the cumulative proportion of surviving patients against survival time; the patients are split into groups according to whether they are p9Ka positive or negative and ErbB2 positive or negative;
  • Figure 4 shows two sets of clonogenic assay data from p9Ka over-expressing cells - the upper graph confirms that cells which over-express p9Ka (the KP1R37 transfectant) are more resistant to taxanes (in this case Paclitaxel) than low p9Ka expressing lines (the RAMA 37 parent), whilst the lower graph shows that when exposed to doxorubicin the low p9Ka cells (RAMA 37) are more resistant than cells which over-express p9Ka (KP1R37);
  • Figure 5 shows comparative drug sensitivities in p9Ka transfectants (KP1R37 cells) - the horizontal line indicates the normalised parental cell drug treatment response, the ascending line indicates taxane resistance and the descending line indicates doxorubicin sensitivity; and
  • Figure 6 shows data from clonogenic cell survival assays for determining the relative drug sensitivity of p9Ka over-expressing and low-p9Ka expressing parental RAMA37 cells - this data shows that p9Ka over-expressing cells are more sensitive to exposure to 5FU than low p9Ka expressing parental cells.
  • the assay of the present invention may be carried out by determining p9Ka alone, or by determining further genes or substances within the subject, and/or within the cancer cells themselves.
  • a further such useful predictive indicator in combination with p9Ka is ErbB2, as explained in more detail below.
  • EGF epithelial growth factor
  • EGFR epithelial growth factor receptor
  • Her2 Another cell surface receptor capable of triggering cell division is Her2 which is 80 % homologous to EGFR but does not bind EGF. Her2 is also called ErbB2 because it is homologous to the avian erythroblastosis viral oncogene v-erbB. Her2 is over-expressed with high frequency in human adenocarcinomas of the breast, stomach, ovary and bladder cancers (Slamon et al, 1987; Yokota et al, 1988; Wright et al, 1990) and in other tumours. In human tumours only over-expression of Her2 occurs. In rats a mutation in Her2 gives rise to the neu oncogene.
  • a form of therapy for Her2 -positive tumours is treatment with the human monoclonal antibody Herceptin.
  • the present invention provides the ability to select the right patients for Herceptin therapy by detecting, in the context of p9Ka expression, the expression or over-expression of Her2/ErbB2, preferably on the surface of the patient's tumour cells. This invention thus provides one of the first clinical medignostic tests in this area.
  • a particularly preferred further predictive indicator is the dominant oncogene, ErbB2.
  • the assay comprises, prior to comparing with a correlation, further determining in the sample the level of expression of ErbB2 or the abundance of ErbB2 protein, and then comparing this level or abundance with a pre-determined correlation between: p9Ka level or p9Ka protein abundance, ErbB2 level or ErbB2 protein abundance, and survival of subjects having cancer; and then determining the prognosis for the subject.
  • the combination of p9Ka determination and ErbB2 determination may, in some instances, correlate in an even more statistically predictive manner with patient survival, than correlations with p9Ka alone.
  • a further aspect of the present invention arises out of the surprising correlation between the p9Ka level and effectiveness of specific chemotherapeutic agents in treating cancer.
  • the present invention also provides a clinical assay for selecting an agent for treating cancer, which assay comprises:
  • the assay of this aspect of the invention may be carried out by determining p9Ka alone, or by determining further genes or substances within the subject, and/or within the cancer cells themselves.
  • a particularly preferred further predictive indicator is the dominant oncogene, ErbB2.
  • the assay comprises, prior to selecting a chemotherapeutic (preferably cytotoxic) agent, further determining in the sample the level of expression of ErbB2 or the abundance of ErbB2 protein.
  • a chemotherapeutic agent preferably cytotoxic
  • the agent comprising a chemotherapeutic agent is selected.
  • the chemotherapeutic agent is preferably selected when p9Ka is over-expressed or p9Ka protein is present at an elevated level.
  • the chemotherapeutic agent is preferably selected if ErbB2 is over-expressed or ErbB2 protein is present at an elevated level.
  • the chemotherapeutic agent is preferably a cytotoxic agent.
  • the chemotherapeutic agent preferably comprises a cytotoxic agent other than a taxane.
  • the chemotherapeutic agent comprises an agent effective in delivering, (either directly or via metabolysis of a precursor substance), 5-fluorouracil (5FU), 5FU derivatives, 5FU pro-drugs, and/or 5FU-related halogenated pyrimidines in the subject. Delivery is preferably in the vicinity ofthe cancer in the subject.
  • the chemotherapeutic agent comprises an anthracycline.
  • the chemotherapeutic agent may comprise doxorubicin, herceptin, tamoxifen, xeloda (capecitabine, a 5FU pro-drug), and/or a platinating agent. It is particularly preferred that when only p9Ka is determined, the chemotherapeutic agent selected comprises doxorubicin. When both p9Ka and ErbB2 are determined, it is preferred that the chemotherapeutic agent selected comprises herceptin. This is because correlation between p9Ka and doxorubicin effectiveness is especially marked, and correlation between p9Ka and herceptin may be marked when ErbB2 is expressed or ErbB2 protein is present.
  • the chemotherapeutic agent may comprise a further anti-cancer agent, such as a chemotherapeutic agent.
  • This agent may be a cytotoxic agent.
  • the further agent preferably comprises an anthracycline, doxorubicin, herceptin, tamoxifen, 5FU, a 5FU derivative, a 5FU pro-drug such as xeloda, a 5FU-related halogenated pyrimidine, a platinating agent and/or a taxane.
  • kit for determining a prognosis for a subject having a cancer which kit comprises:
  • kit for selecting an agent for treating a cancer, which kit comprises:
  • kits may be employed to perform the methods of the present invention.
  • the means for determining the level of expression of p9Ka or the abundance of p9Ka protein is not especially limited, but preferably is a probe for p9Ka mRNA or a labelled antibody against ⁇ 9Ka protein.
  • the means for determining the prognosis is not especially limited, but it is preferred that this means comprises a pre-determined correlation between p9Ka level or p9Ka protein abundance and survival of subjects having cancer. Such a correlation is generally a correlation as described in detail above in relation the methods of the present invention.
  • the means for selecting the agent is also not especially limited, but it is preferred that this means comprises a pre-determined correlation between p9Ka level or p9Ka protein abundance and sensitivity of the cells to the agent for treating cancer that is being tested. This latter correlation in the context of the present invention may be a relationship between either the sensitivity or the resistance of the cells to the anti-cancer agent, and the level of p9Ka or p9Ka protein in the cancer cells.
  • the sensitivity or resistance of the cells to the anti-cancer agent may be measured using conventional methods, e.g. using a clonogenic assay.
  • Some extremely useful and predictive correlations have been provided by the present inventors using clonogenic assays, and further correlations can be provided, if desired, using this method.
  • Types of correlation are not especially limited. Thus correlations may relate to specific types of subject, limited (for example) by age or sex, and correlations may relate to specific classes of cancer, such as metastatic cancers and/or solid tumours, or to specific cancers, such as breast cancer.
  • a correlation or relationship may, if desired, be provided in the form of a table, graph or chart which numerically, graphically or otherwise (e.g. in words) sets out the relationship in a manner suitable for comparing experimental results with the table or chart to make a choice or draw a conclusion.
  • the kit for selecting an agent for treating cancer further comprises a chemotherapeutic agent, such as doxorubicin, for treating a subject.
  • a chemotherapeutic agent such as doxorubicin
  • the kit may, in addition to the chemotherapeutic agent, comprise one or more additional agents for treating cancer. These agents are preferably platinating agents, such as cis-platin, and/or taxanes, such as taxol.
  • the present invention has the advantage of being able to offer treatment such as adjuvant chemotherapy to slow down the growth of, or even eradicate, the relatively small numbers of metastatic cells already present in such patients. It is especially useful if the treatment is with cytotoxic drugs already in established use in the clinic. In order to decide which cytotoxic drug to use it is necessary to know that the expression or over-expression of the metastagene does not make cells more resistant to treatment with that drug. The particular cytotoxic drug is then likely to work effectively in an adjuvant situation.
  • non-neoplastic cells present in carcinomas e.g. some lymphocytes endotheleal cells and fibroblasts, also contain p9Ka (Gibbs et al, 1995) and they can complicate the overall interpretation.
  • measurement of p9Ka can be carried out using polyclonal antibodies or monoclonal antibodies that react with the p9Ka protein or mRNA.
  • DNA, RNA or protein arrays or microarrays may be employed in the present invention, as may chips.
  • the level of expression of p9Ka is not especially limited in the present mvention, provided that p9Ka is detectably expressed, or p9Ka protein is detectably present.
  • 1% or more of the carcinoma cells may be immunocytochemically stainable by antibodies to p9Ka, or 5% or more, 10% or more, 25% or more or 50% or more, depending on the patient and the cancer cells involved.
  • p9Ka is over-expressed. Treatment with, for example, doxorubicin is particularly effective if p9Ka is over- expressed.
  • this translates into 1% or more of the carcinoma cells being immunocytochemically stainable by antibodies to p9Ka in experimental animal systems (Davies et al, 1993; 1996) and in human breast cancer specimens (Barraclough et al., 1984; Davies et al, 1993).
  • This immunocytochemical cut-off point can be determined according to the judgement of the individual carrying out the present method, depending on the particular cancer cells and patient involved.
  • the cut-off point for ErbB2, and other genetic indicators, is generally considered to be the same or similar to that of p9Ka.
  • a clinical test may be developed, e.g. for doxorubicin sensitivity, based on the measurement of in situ p9Ka protein expression typically by standard techniques of immunocytochemistry in a research environment, but for diagnostic purposes cheaper and more rapid methods are preferable.
  • the sample may be a sample of cells or may be a cell extract.
  • the cells are pre-cancerous cells, or are tumour cells. It is generally preferred that the cells are extracted from the subject before undergoing the present method.
  • the subject is generally a human subject known or suspected to be suffering from a cancer. Of particular interest are solid tumours, such as breast cancer, prostate cancer, lung cancer, bowel cancer, malignant melanoma, ovarian cancer, head cancer, neck cancer, and uro-genital cancers such as bladder cancer.
  • the present invention is particularly effective in providing a prognosis for and selecting treatment for cancers which readily metastasise. The present methods thus allow for accurate patient stratification.
  • mRNA levels for p9Ka follow the same pattern as for the p9Ka protein (in particular in mammary tissue cultured cell lines) this makes it possible to carry out a functional assay, e.g. for prognosis or resistance to doxorubicin, by in situ hybridisation for p9Ka mRNA from tumour samples, and use this to determine the relative percentages of carcinoma cells expressing p9Ka mRNA.
  • a functional assay e.g. for prognosis or resistance to doxorubicin
  • mRNA levels for p9Ka need to be assessed in the carcinoma cells, this may be accomplished by first laser micro-dissecting the carcinoma cells away from host contaminating tissue (Schutze et al., 1998)) or by separation in a FACS machine (see below) and then using competitive Reverse Transcriptese PCR for estimation of p9Ka mRNA (Taylor et al, 1998).
  • the immunocytochemical test outlined above can be automated and improved using confocal laser fluorescence microscopy or other related laser cytometric methods.
  • a scanning system is used such as those described in PCT/US91/09217 and PCT/US95/01886.
  • the microscopy system is also able to analyse multiple fluorescent dyes.
  • antibodies against p9Ka are labelled with a dye.
  • Antibodies exist against a number of known forms of the p9Ka protein.
  • a diagnostic test may comprise the steps of:
  • micro-dissecting preferably laser micro-dissecting
  • Over-express or show elevated levels of p9Ka i.e. are labelled with at least a threshold quantity of antibody against p9Ka.
  • Fluorescence Activated Cell Sorting separates cells in a single-cell suspension in a manner dependent on the cells being labelled with a fluorescent marker (Begg et al, 1985).
  • Micro-dissection e.g. laser micro-dissection
  • keratins e.g. keratin 18 or 19 can be used to separate the epithelial cells from host cells (Rudland et al, 1993).
  • the p9Ka and keratin antigens occur in the cytoplasm of cells and hence cells may require permeabilisation e.g. by treatment with methanol etc.
  • Multi-channel fluorescent detection devices have been constructed that can separate cells on the basis of labelling with multiple different fluorescent labels. These have multiple lasers which can excite fluorescence at different frequencies and the detector will detect different emission frequencies. The same labelled probes as those described above for use in a confocal scanning fluorescence microscope would be appropriate.
  • a diagnostic test may comprise the steps of:
  • micro-dissecting e.g. laser micro-dissecting
  • material to separate normal tissue from tumour material e.g. laser micro-dissecting
  • the present invention provides a method for identifying an agent effective in treating a cancer, which cancer is associated with p9Ka expression or with the presence of p9Ka protein, which method comprises measuring the sensitivity to the agent of cancer cells that express p9Ka or in which p9Ka protein is present.
  • This method may thus be a method for screening for agents which will be effective anti-cancer agents.
  • a cancer which is associated with p9Ka expression or with the presence of p9Ka protein is a cancer comprising cells which express p9Ka or in which p9Ka protein is present.
  • measuring the resistance of the cells is intended to be included by the phrase 'measuring the sensitivity' ofthe cells.
  • This method provides a useful route for identifying further agents or substances (in particular cytotoxic anti-cancer agents) which are more effective in treating cancers in which p9Ka is expressed or in which p9Ka protein is present, than cancers in which p9Ka is not expressed or is absent.
  • This aspect of the present invention also provides a kit for identifying a substance effective in treating a cancer, which cancer is associated with p9Ka expression or with the presence of p9Ka protein, which kit comprises:
  • cancer cells that express p9Ka or in which p9Ka protein is present; and (ii) a means for determining the sensitivity of the cells to the substance.
  • the cancer is not especially limited, but is preferably a metastatic cancer.
  • a metastatic cancer is a cancer which metastasises, and is likely to be associated with a poor prognosis for the patient.
  • Cancers of particular interest in the present invention are solid tumours, such as breast cancer, prostate cancer, lung cancer, bowel cancer, malignant melanoma, a sarcoma, testicular cancer, ovarian cancer, head cancer, neck cancer, and uro-genital cancers such as bladder cancer.
  • the cancer cells, or extracts therefrom, used in the methods and kits of this aspect of the invention are not especially limited, provided that they express p9Ka or contain p9Ka protein.
  • the cancer cells may be cell lines derived from any metastatic cancer such as the specific cancers mentioned above.
  • a cell line of particular utility is the RAMA 37-p9Ka (KP1R 37) cell line and cells derived from this cell line.
  • Cells which are also preferred for use in this aspect of the invention are rat cells comprising rat p9Ka, rat cells comprising human p9Ka or human cells comprising human p9Ka.
  • RAMA 37-p9Ka is a p9Ka transfected benign rat breast cancer cell line.
  • Cell lines can be derived from this cell line by implanting this cell line in rats and harvesting metastatic cells that have spread from the primary tumour to the animal's lungs or lymph nodes and which are grown again as in vitro cell lines.
  • New cell lines can be constructed in which human p9Ka is expressed in benign human cell lines and metastatic variants of this line, obtained by growth in immunosuppressed animals and harvesting cells from metastases.
  • the method for determining the sensitivity of the cells to the substance is not especially limited. However, a clonogenic assay is the preferred method for carrying out this determination.
  • a means for determining the level of expression of p9Ka or the abundance of p9Ka protein for selecting an agent for the treatment of cancer.
  • the means for determining the level of expression of p9Ka or the abundance of p9Ka protein is not especially limited, and is preferably any one of such means already described above.
  • this aspect of the present invention further comprises using a means for determining the level of expression of ErbB2 or the abundance of ErbB2 protein, for selecting an agent for the treatment of cancer.
  • This latter means is also not especially limited and may be as discussed above in respect of the methods of the present invention.
  • the present invention also involves a method of treating cancer in a subject having cancer cells in which p9Ka is expressed or p9Ka protein is present.
  • This aspect of the invention provides use of an therapeutic agent in the manufacture of a medicament effective in treating cancer in a subject having cancer cells in which p9Ka is expressed or p9Ka protein is present.
  • the subject has cancer cells in which ErbB2 is expressed or ErbB2 protein is present.
  • the therapeutic agent selected is not particularly limited, provided that it is effective in treating p9Ka expressing cancer, although typically the therapeutic agent is a chemotherapeutic agent.
  • the medicament comprises doxorubicin, herceptin, or related compounds.
  • the therapeutic agent is one that is capable of preventing, reducing or inhibiting metastasis, e.g. by preventing, reducing or inhibiting production of p9Ka protein, or the activity of p9Ka protein.
  • the type of cancer is not especially limited.
  • the cancer is preferably cancer in a mammal, such as humans.
  • the cancer is typically breast cancer, prostate cancer, lung cancer, bowel cancer, malignant melanoma, a sarcoma, testicular cancer, ovarian cancer, head cancer, neck cancer, or a uro-genital cancer. It is particularly preferred that the cancer is breast cancer.
  • the present invention will be described in further detail by way of example only with reference to the following specific embodiments.
  • Example 1 - p9Ka as a prognostic indicator
  • the cell nuclei were counterstained blue by Meyers Haemalum (Warburton et al, 1982).
  • the percentage of carcinoma cells stained for p9Ka was determined from two sections of each specimen, 5 to 10 fields per section (250x magnification) (about 200-400 cells) in a microscope by two independent observers. Those patients who died of causes other than cancer were eliminated from the study (62). The remaining 287 patients were divided into two groups in graph A of Figure 1; 107 with ⁇ 1% of the carcinoma cells stained for p9Ka (dotted line, no p9Ka expression, -) and 180 with >1% of the carcinoma cells stained for p9Ka (full line, over-expressed p9Ka, +).
  • Relative risk for survival (RR) and 95 % confidence interval (CI) were determined using a Cox univariate analysis with one degree of freedom.
  • lymph node metastasis previously one of the strongest predictors of prognosis, need no longer be tested for now that p9Ka is available.
  • p9Ka p9Ka
  • Table 1 thus demonstrates the surprising utility of the present invention by identifying p9Ka as the unique optimal prognostic indicator in cancer, especially breast cancer, which supplants all previous prognostic indicators.
  • Example 4 The combined use ofp9Ka expression and ErbB2/Her2 expression to identify breast cancer patients who should be treated with Herceptin as adjuvant chemotherapy. Tests were carried out to demonstrate the utility of the combined predictive utility of p9Ka expression and ErbB2 expression.
  • Figure 3 demonstrates that expression or over-expression of the ErbB2 protein generally carries a prognostic significance when p9Ka is also expressed or over-expressed.
  • the present invention now enables measuring a combination of p9Ka and ErbB2 over-expression, to provide an improved and more exact medignostic test for determining which patients with cancer, such as breast cancer, should be treated with Herceptin.
  • a combination of over-expression of both p9Ka and ErbB2/HER2 is shown by the present invention to be an indication for the use of Herceptin, preferably as an adjuvant treatment in patients with early breast cancer at high risk of developing metastases.
  • Example 5 The use ofp9Ka protein expression to indicate that taxanes (as compared to anthracyclines) are less likely to be useful chemotherapy agents against breast cancers (or other tumours) that over-express p9Ka.
  • Tests were carried out to demonstrate the utility of measuring p9Ka expression levels to determine which type of chemotherapeutic treatment to give to specific patients.
  • Cell survival on exposure to the drugs was determined using conventional clonogenic assays. The results are shown in Figure 4 and Figure 5.
  • p9Ka expression can provide a specific medignostic test which can enable a choice to be made between different cytotoxic drugs as to drug which is most likely to be effective in p9Ka over-expressing cells.
  • the present invention generally provides a clinical assay for selection of a chemotherapeutic agent, especially for selecting a non-taxane such as an anthracycline, for treating a cancer whose cells express or over-express p9Ka.
  • the present invention also generally provides a clinical assay for selection of a chemotherapeutic agent, especially for selecting a taxane such as Paclitaxel, for treating a cancer whose cells do not express p9Ka.
  • Example 6 The use of medignostic tests for p9Ka over-expression to indicate that the cytotoxic drug 5-Fluorouracil (5FU) is likely to be of benefit to patients whose breast cancers over-express p9Ka.
  • Tests were carried out to demonstrate the utility of measuring p9Ka expression levels to determine further types of chemotherapeutic treatment to administer to specific patients.
  • Cell survival on exposure to 5FU was investigated, and determined using conventional clonogenic assays. The results are shown in Figure 6.
  • Figure 6 is similar to the earlier Figures of data from clonogenic cell survival assays to determine the relative drug sensitivity of cells over-expressing p9Ka and cells not expressing p9Ka.
  • RAMA 37 parental cells and KP1R 37 transfectants were employed, as in the above tests.
  • p9Ka over-expressing cells are confirmed as being more sensitive to exposure to 5FU than low p9Ka expressing parental cells.
  • This result confirms that p9Ka over-expression can provide a medignostic test indicating which patients should receive 5FU treatment for advanced disease or as adjuvant therapy.
  • a medignostic test is also provided indicating which patients should receive prodrugs whose active product is 5FU generated at the tumour site - for example the drug Xeloda.

Abstract

L'invention concerne un procédé de dosage clinique permettant de prévoir l'évolution du cancer d'un sujet. Ce procédé consiste à (a) déterminer, dans un échantillon comprenant une ou plusieurs cellules ou un extrait de ces dernières, le niveau d'expression de p9Ka ou la quantité des protéines p9Ka; (b) comparer le niveau ou la quantité déterminée dans l'étape (a) avec une corrélation prédéterminée entre le niveau p9Ka ou la quantité de protéines p9KA et la survie de sujets souffrant d'un cancer; et (c) prévoir l'évolution du cancer en se fondant sur ces comparaisons. L'invention traite, en outre, d'un procédé de sélection d'un agent de traitement du cancer. Ce procédé consiste à (a) déterminer, dans un échantillon comprenant une ou plusieurs cellules ou un extrait de ces dernières, le niveau d'expression de p9Ka ou la quantité de protéines p9Ka et (b) lors de l'expression de p9Ka, et/ou lors de la présence de la protéine p9Ka, à sélectionner un agent comprenant un agent chimiothérapeutique pour traiter le cancer d'un sujet.
PCT/GB2000/001866 1999-05-14 2000-05-15 Traitement du cancer WO2000070352A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU45981/00A AU4598100A (en) 1999-05-14 2000-05-15 Treating cancer

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB9911302.9 1999-05-14
GBGB9911302.9A GB9911302D0 (en) 1999-05-14 1999-05-14 Treating cancer
GBGB9914971.8A GB9914971D0 (en) 1999-05-14 1999-06-25 Treating cancer
GB9914971.8 1999-06-25
GB0007254.6 2000-03-24
GB0007254A GB0007254D0 (en) 1999-05-14 2000-03-24 Treating cancer

Publications (1)

Publication Number Publication Date
WO2000070352A1 true WO2000070352A1 (fr) 2000-11-23

Family

ID=27255622

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2000/001866 WO2000070352A1 (fr) 1999-05-14 2000-05-15 Traitement du cancer

Country Status (2)

Country Link
AU (1) AU4598100A (fr)
WO (1) WO2000070352A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010132958A1 (fr) * 2009-05-22 2010-11-25 Garvan Institute Of Medical Research Procédés pour prédire la réactivité à un traitement
US9683032B2 (en) 2012-10-30 2017-06-20 Cancer Research Technology Limited Anti-S100A4 antibody molecules and their uses

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ALBERTAZZI E. ET AL.: "Expression of metastasis-associated genes h-mts1 (S100A4) and nm23 in carcinoma of breast is related to disease progression", DNA AND CELL BIOLOGY, vol. 17, no. 4, 1998, pages 335 - 342, XP000937972 *
BÖNI R. ET AL.: "Immunohistochemical localization of the Ca2+ binding S100 proteins in normal human skin and melanocytic lesions", BRITISH JOURNAL OF DERMATOLOGY, vol. 137, 1997, pages 39 - 43, XP000951528 *
KERNOHAM N.M. ET AL.: "S-100 protein: a prognostic indicator in cutaneous malignant melanoma?", HISTOPATHOLOGY, vol. 11, 1987, pages 1285 - 1293, XP000949364 *
LLOYD B.H. ET AL.: "Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumor cells", ONCOGENE, vol. 17, 1998, pages 465 - 473, XP000938007 *
RUDLAND P.S. ET AL.: "Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer", CANCER RESEARCH, vol. 60, 15 March 2000 (2000-03-15), pages 1595 - 1603, XP000939339 *
SHRESTHA P. ET AL.: "Localization of Ca2+-binding S100 proteins in epithelial tumours of the skin", VIRCOWS ARCH, vol. 432, 1998, pages 53 - 59, XP000951590 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010132958A1 (fr) * 2009-05-22 2010-11-25 Garvan Institute Of Medical Research Procédés pour prédire la réactivité à un traitement
US9683032B2 (en) 2012-10-30 2017-06-20 Cancer Research Technology Limited Anti-S100A4 antibody molecules and their uses

Also Published As

Publication number Publication date
AU4598100A (en) 2000-12-05

Similar Documents

Publication Publication Date Title
Rudland et al. Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer
Lark et al. Overexpression of focal adhesion kinase in primary colorectal carcinomas and colorectal liver metastases: immunohistochemistry and real-time PCR analyses
Blanchard et al. Claudins 1, 3, and 4 protein expression in ER negative breast cancer correlates with markers of the basal phenotype
Reimer et al. FasL: Fas ratio–a prognostic factor in breast carcinomas
Lu et al. Increased expression of δ-catenin/neural plakophilin-related armadillo protein is associated with the down-regulation and redistribution of E-cadherin and p120ctn in human prostate cancer
Jesinghaus et al. Increased intraepithelial CD3+ T-lymphocytes and high PD-L1 expression on tumor cells are associated with a favorable prognosis in esophageal squamous cell carcinoma and allow prognostic immunogenic subgrouping
Laurell et al. Identification of biomarkers of human pancreatic adenocarcinomas by expression profiling and validation with gene expression analysis in endoscopic ultrasound-guided fine needle aspiration samples
AU676720B2 (en) Method of detecting carcinoma
CN101027412A (zh) 用于膀胱癌检测的尿标记物
US20020132274A1 (en) Diagnostic and monitorings methods for cancer
WO2006062118A1 (fr) Nouveaux marqueurs pour le pronostic predictif du carcinome papillaire de la thyroide
US20100068717A1 (en) Cancer therapy prognosis
CN102667472A (zh) Tivozanib应答预测
US20080166733A1 (en) Method for determining the prognosis of cancer patients by measuring levels of bag expression
Radulović et al. Immunohistochemical expression of NEDD9, E-cadherin and γ-catenin and their prognostic significance in pancreatic ductal adenocarcinoma (PDAC)
Kondo et al. Detection of KK-LC-1 protein, a cancer/testis antigen, in patients with breast cancer
Andersson et al. HER-2/neu (c-erbB-2) evaluation in primary breast carcinoma by fluorescent in situ hybridization and immunohistochemistry with special focus on intratumor heterogeneity and comparison of invasive and in situ components
Kotsinas et al. Proliferation, but not apoptosis, is associated with distinct β-catenin expression patterns in non-small-cell lung carcinomas: relationship with adenomatous polyposis coli and G1-to S-phase cell-cycle regulators
Keene et al. The clinical significance of micrometastases and molecular metastases
Fabjani et al. HMGA2 is associated with invasiveness but not a suitable marker for the detection of circulating tumor cells in breast cancer
US20100152058A1 (en) Cancer markers
US10247732B2 (en) Materials and methods for diagnosing and predicting the course of prostate cancer
Orlando et al. Are all high-grade breast cancers with no steroid receptor hormone expression alike? The special case of the medullary phenotype
Huang et al. The expression of novel gene URG4 in osteosarcoma correlation with patients' prognosis
Tong et al. Messenger RNA determination of estrogen receptor, progesterone receptor, pS2, and plasminogen activator inhibitor-1 by competitive reverse transcription-polymerase chain reaction in human breast cancer

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

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

Ref country code: JP