EP2212433A2

EP2212433A2 - Ptpl1 as a biomaker of survival in breast cancer.

Info

Publication number: EP2212433A2
Application number: EP08805154A
Authority: EP
Inventors: Françoise VIGNON; Carole Puech; Gilles Freiss
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM
Priority date: 2007-10-08
Filing date: 2008-10-08
Publication date: 2010-08-04
Also published as: EP2048244A1; WO2009047274A2; WO2009047274A3

Abstract

The present invention relates to the use of PTPL1 as a biomarker of survival in breast cancer. More particularly, the present invention relates to a method for predicting the survival of a breast cancer patient comprising determining the expression level of the PTPL1 gene in a biological sample obtained from said patient.

Description

PTPL1 AS A BIOMAKER OF SURVIVAL IN BREAST CANCER

FIELD OF THE INVENTION

The present invention relates to the use of PTPL1 as a biomarker of survival in breast cancer.

BACKGROUND OF THE INVENTION

Breast cancer affects about one million times per year, and represents the most common form of cancer in females, affecting approximately 10% of all women at some stage of their life in the Western world. Men can also develop breast cancer, although their risk is less than 1 in 1000. Breast cancer risk is modified by many different factors. Besides gender, the main risk factors of getting breast cancer are increasing age, having a family history of breast cancer and exposure to endogenous or exogenous estrogens. Actually, factors contributing to an increased cumulative estrogen exposure have clearly been shown to increase the risk of breast cancer development. In a first aspect, it is actually necessary to determine the histological features of the breast cancer of a patient. In fact, there is a great diversity of tumours characteristics in breast cancer. Different makers have been associated with specific breast cancer types. As an example of such markers, one can cite markers associated to proliferation (Ki-67) and cell cycle (cyclin E, cyclin D1 ) (Esteva FJ. et al. 2004), to mitogenic and survival pathways

(tyrosine kinase receptors HERs) (Pawlowski V. et al. 2000) and to invasion process (uPA, Cathepsin D) (Peyrat JP. et al. 1998; Thorpe SM et al 19989).

To illustrate this diversity, it is known that tumours not expressing ER are treated aggressively such as with immediate chemotherapy after surgical intervention, because of the aggressiveness of such breast tumour compared to ER positive breast cancers.

Despite efforts to improve treatment and management of cancer patients, survival in cancer patients has not improved over the past two decades for many cancer types. Optimal therapy will be based on a combination of diagnostic and prognostic information. An accurate and reproducible diagnostic test is needed to provide a prognostic assessment that will provide specific information regarding survival. In breast cancer, the clinical and biological variables commonly used to predict the survival of primary chirurgical treatments are regional lymph nodes invasion, histological grade, and hormone receptor expression. All these parameters are well recognized prognostic and predictive factors.

Nevertheless, these variables do not enable to establish a specific and complete survival prognostic. In fact, there is an important heterogeneity between patients suffering from the same breast cancer type.

Thus, there is an existing need to provide further biomarkers that will strengthen the prediction of survival in breast cancer patients. The inventors have now discovered that the specific analysis of PTPL1 could provide a prognostic of survival in breast cancer patients, independently of the breast cancer types.

The patent application WO 2004/079014 concerns breast cancer typing and most especially breast cancer expressing oestrogen receptor typing. As such, this application discloses a gene signature comprising several hundred of genes including PTPN13, for identifying in breast biopsies those corresponding to the breast cancer type expressing oestrogen receptor.

SUMMARY OF THE INVENTION The present invention relates to a method for predicting the survival of a breast cancer patient comprising determining the expression level of the PTPL1 gene in a biological sample obtained from said patient.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The term "PTPL1 " has its general meaning in the art and refers to the protein tyrosine phosphatase PTPL1 . PTPL1 is also known as hPTP1 E, PTP- BAS, FAP-1 , or PTPN13. PTPL1 can be from any source, but typically is a mammalian (e.g., human and non-human primate) PTPL1 , particularly a human PTPL1 . The "term PTPL1 gene" refers to any nucleotide sequence encoding the PTPL1 mRNA and protein, such as a genomic DNA sequence and any naturally occurring PTPL1 and variants and modified forms thereof. It can also encompass artificial sequences such as cDNA encoding the PTPL1 mRNA and protein. Exemplary human native PTPL1 nucleotide sequences are provided in GenBank database under accession numbers NC_000004 for PTPL1 gene, NM_080683 (SEQ ID NO:1 ), NM_080684 (SEQ ID NO:2), NM_080685 (SEQ ID NO:3) and NM_006264 (SEQ ID NO:4) for PTL1 mRNA (cDNA). The term "PTPL1 mRNA" has its general meaning in the art and refers to the messenger RNA which is synthesized upon expression of the PTPL1 gene. The term "PTPL1 protein" refers to the amino acid sequence resulting from the expression of the PTPL1 gene, and any naturally occurring PTPL1 and variants and modified forms thereof. Exemplary human native PTPL1 amino acid sequences are provided in GenPept database under accession numbers, NP_542414 (SEQ ID NO:5), NP_542415 (SEQ ID NO:6), NP_542416 (SEQ ID NO:7) and NP_006255 (SEQ ID NO:8).

The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA. Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, myristilation, and glycosylation.

The term "anti-PTPL1 antibody" refers to an antibody or a fragment thereof which recognizes PTPL1 .

As used herein, the term "predetermined value" refers to the amount of PTPL1 in biological samples obtained from the general population or from a selected population of subjects. For example, the predetermined value may be of the amount of PTPL1 obtained from breast cancer patients who have survived. The predetermined value can be a threshold value, or a range. The predetermined value can be established based upon comparative measurements between breast cancer patients who have survived and breast cancer patients who have not survived.

The term "patient" as used herein denotes a mammal such as a rodent, a feline, a canine and a primate. Preferably, a patient according to the invention is a human. The term "breast cancer patient" denotes a patient who is affected with a breast cancer.

The patient suffers from a specific breast cancer type.

As an example, the patient can be an oestrogen receptor (ER)-positive breast cancer patient or an oestrogen receptor (ER)-negative breast cancer patient.

In a particular embodiment the patient is an oestrogen receptor (ER)- positive breast cancer patient.

As used herein, the expression "oestrogen receptor (ER)-positive breast cancer patient" or "oestrogen receptor (ER)-negative breast cancer patient" means that said breast cancer patient express or not express oestrogen receptor at the surface of its cells respectively. The expression of oestrogen receptor can be simply determined by well known methods such as immunochemistry or PCR.

The term "biological sample" means any biological sample derived from a patient. As an example, said biological sample refers to a breast biopsy which has been removed from the body of a patient prior to executing the method of the invention, or to a cell or cell line obtained from such a breast biopsy.

The term "biomarker", as used herein, refers generally to a molecule, i.e., a gene (or nucleic acid encoding said gene), protein, the expression of which in a biological sample from a patient can be detected by standard methods in the art (as well as those disclosed herein), and is predictive or denotes a condition of the subject from which it was obtained.

Predictive methods of the invention:

The present invention relates to a method for predicting the survival of a breast cancer patient comprising/consisting of determining the expression level of the PTPL1 gene in a biological sample obtained from said patient.

Preferably, said method is an in vitro method.

The biological sample may contain nucleic acids or proteins. Examples of such biological samples include tissues, cell samples, organs, biopsies, etc. The biological sample may result from a biopsy, and more specifically from a breast biopsy. Breast cancer biopsy is a well known technique in the art, and may involve the use of a needle.

Total nucleic acids or proteins can be easily extracted from cell or tissue samples. The biological sample may be treated prior to its use, e.g. in order to render nucleic acids or proteins available. Techniques of cell or protein lysis, concentration or dilution of nucleic acids, are known by the skilled person. In one embodiment, the invention relates to a method for predicting the survival of breast cancer patient comprising/consisting in determining the quantity of mRNA encoding PTPL1 in a cell or tissue sample obtained from said patient. Determination of the expression level of a gene can be performed by a variety of techniques. Generally, the expression level as determined is a relative expression level.

More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount of nucleic acids of interest originally in the sample.

In a preferred embodiment, the expression level may be determined by measuring the quantity of mRNA in a biological sample of said patient.

Advantageously, the method of the invention further comprises a step of comparing the concentration of the PTPL1 mRNA with a predetermined value. Said comparison is indicative of the survival for the breast cancer patient, with an overexpression of the PTPL1 mRNA in the biological sample compared to the predetermined value being associated with a longer survival prognostic. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). In a preferred embodiment, the expression level of the PTPL1 gene is determined by RT-PCR, preferably quantitative or semi-quantitative RT-PCR, even more preferably real-time quantitative or semi-quantitative RT-PCR. Other methods of amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCI, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In another embodiment, the invention relates to a method for predicting the survival of a breast cancer patient comprising/consisting in measuring the quantity of the PTPL1 protein in a biological sample obtained from said patient. In a particular embodiment, the methods of the invention comprise contacting the biological sample with a binding partner capable of selectively interacting with the PTPL1 protein present in the biological sample.

The binding partner may be an antibody that may be polyclonal or monoclonal, preferably monoclonal. In another embodiment, the binding partner may be an aptamer.

Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.

Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally described by Kohler and Milstein (1975); the human B- cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).

Alternatively, techniques described for the production of single chain antibodies (see e.g. U.S. Pat. No. 4,946,778) can be adapted to produce anti-PTPL1 , single chain antibodies. Antibodies useful in practicing the present invention also include anti-PTPL1 fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and/or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to PTPL1 . For example, phage display of antibodies may be used. In such a method, single-chain Fv (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, e. g., M13. Briefly, spleen cells of a suitable host, e. g., mouse, that has been immunized with a protein are removed. The coding regions of the VL and VH chains are obtained from those cells that are producing the desired antibody against the protein. These coding regions are then fused to a terminus of a phage sequence. Once the phage is inserted into a suitable carrier, e. g., bacteria, the phage displays the antibody fragment. Phage display of antibodies may also be provided by combinatorial methods known to those skilled in the art. Antibody fragments displayed by a phage may then be used as part of an immunoassay.

Examples of commercially available monoclonal antibodies for PTPL1 include those obtained from Leica Microsystems under the reference AC21 .

Examples of commercially polyclonal antibodies for PTPL1 include those obtained from Santa Cruz Biotech under the references sc-15356, sc- 1 138, or sc-1729; from R&D systems under the reference ref AF3577; or from Abeam under the references Ab26218 and Ab 31017. In another embodiment, the binding partner may be an aptamer.

Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. 1997. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S. D., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A, that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

The binding partners of the invention such as antibodies or aptamers, may be labelled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal.

As used herein, the term "labelled", with regard to the antibody, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or lndocyanine (Cy5)) to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. For example radioactive molecules include but are not limited radioactive atom for scintigraphic studies such as 1123, 1124, In1 1 1 , Re186, Re188.

The aforementioned assays may involve the binding of the binding partner (ie. Antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

PTPL1 protein may be measured by using standard immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, agglutination tests; enzyme-labelled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of anti-PTPL1 antibodies. A biological sample containing or suspected of containing PTPL1 is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

Alternatively an immunohistochemistry (IHC) method may be used. IHC specifically provides a method of detecting targets in a sample or tissue specimen in situ. The overall cellular integrity of the sample is maintained in IHC, thus allowing detection of both the presence and location of the targets of interest. Typically a sample is fixed with formalin, embedded in paraffin and cut into sections for staining and subsequent inspection by light microscopy. Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling. Examples of known IHC systems include, for example, EnVision(TM) (DakoCytomation), Powervision(R) (Immunovision, Springdale, AZ), the NBA(TM) kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine(R) (Nichirei Corp, Tokyo, Japan).

In particular embodiment, a tissue section (ie a breast tissue sample) may be mounted on a slide or other support after incubation with anti-PTPL1 antibodies. Then, microscopic inspections in the sample mounted on a suitable solid support may be performed. For the production of photomicrographs, sections comprising samples may be mounted on a glass slide or other planar support, to highlight by selective staining the presence of PTPL1 protein.

Therefore IHC samples may include, for instance: (a) preparations comprising fresh tissues or cells isolated form said tissues (b) fixed and embedded said tissue or cells samples and (c) detecting PTPL1 protein in said tissues or cells samples. In some embodiments, an IHC staining procedure may comprise steps such as: cutting and trimming tissue, fixation, dehydration, paraffin infiltration, cutting in thin sections, mounting onto glass slides, baking, deparaffination, rehydration, antigen retrieval, blocking steps, applying primary anti-PTPL1 antibody, washing, applying secondary antibody (optionally coupled to a suitable detectable label), washing, counter staining, and microscopic examination.

Measuring the PTPL1 protein (with or without immunoassay-based methods) may also include separation of the compounds: centrifugation based on the compound's molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used. Once separated, PTPL1 may be identified based on the known "separation profile" e. g., retention time, for that compound and measured using standard techniques.

Alternatively, the separated compounds may be detected and measured by, for example, a mass spectrometer.

In one embodiment, the method of the invention further may comprise a step of comparing the concentration of the PTPL1 protein with a predetermined value. Said comparison is indicative of the survival in the breast cancer patient, with an overexpression of the PTPL1 protein in the biological sample compared to the predetermined value being associated with a longer survival prognostic.

Preferably, the predetermined value of the PTPL1 is of 5.7 fold that of the PTPL1 expression on MCF-7 cells. MCF-7 cells are a well-characterized oestrogen receptor (ER) positive control cell line (cells are positive for cytoplasmic oestrogen receptors).

In a particular embodiment, the method according to the present invention is also a method for typing the breast cancer and said method further comprises the steps of: a) analysing a breast cancer marker chosen in the group comprising oestrogen and progesterone receptor expression, erb-2/HER2 expression, Ki-67, cyclin E, cyclin D1 , uPA, cathepsin D, or analysing a parameter selected in the group comprising regional lymph nodes invasion analysis, histological grade analysis, and the size of the tumour , and b) typing said breast cancer. Preferably, said marker is chosen in the group consisting in oestrogen and progesterone receptor expression, erb-2/HER2 expression, Ki-67, cyclin E, cyclin D1 , uPA, cathepsin D.

Preferably, the parameter is selected in the group consisting in regional lymph nodes invasion analysis, histological grade analysis, and the size of the tumour.

Another object of the invention is PTPL1 as a biomarker of survival in breast cancer.

The method of the invention may be thus useful for classifying breast cancer patients and then may be used to choose the accurate treatment. For example, patients with a low score of survival may receive a more intensive treatment and attention compared to patient with a high score. Such method may thus help the physician to make a choice on a therapeutic treatment which can accordingly consist in administering accurate drugs to the patients.

Particularly, the method according to the present invention as previously described is a method for predicting the survival of a patient suffering from breast cancer, wherein said breast cancer is an oestrogen receptor (ER)-positive breast cancer.

Yet another object of the invention relates to a kit for predicting the survival of a breast cancer patient, comprising means for measuring the expression of PTPL1 gene in a biological sample obtained from the sample. The kit may include probes and primers as above described. The kit may include an antibody, or a set of antibodies as above described. In a particular embodiment, the antibody or set of antibodies are labelled as above described. The kit may also contain other suitably packaged reagents and materials needed for the particular detection protocol, including solid-phase matrices, if applicable, and standards.

The invention will further be illustrated in view of the figures and example.

FIGURE LEGENDS

Figure 1 : Kaplan-Meier Estimates of Overall Survival for all 291 Patients. The clinical cutoff value corresponds to the upper quartile.

Figure 2: Kaplan-Meier Estimates of Overall Survival and relapse free survival for the 155 Node positive Patients. The clinical cutoff value corresponds to the upper quartile.

Figure 3: Kaplan-Meier Estimates of Overall Survival for the ER+ subgroup of patients. The clinical cut-off value corresponds to the upper quartile. Numbers in parentheses indicate failures/total number of patients in each group.

EXAMPLE

Material and Methods :

Patients: The pilot study involved 59 breast tumour samples; the mean age of the patients was 59.7 years (range 35-81 ). The median duration follow-up of living patients was 7.5 years. The number of deaths was 21 , and the number of relapses was 27.

To confirm the results in a larger sample set, 232 complementary breast tumours samples were included, constituting a testing set of 291 patients (Table 1 ). AII patients underwent surgery for locoregional disease in the Centre Oscar Lambret, the anticancer center of the north of France, between May 1989 and December 1991 . The mean age of the patients was 57.9 years (range 26-90). Patients were treated by segmentectomy when the tumour was smaller than 3 cm wide and by total mastectomy if the tumour was larger or centrally located. Surgery was followed by radiation therapy. Node-positive premenopausal patients, ER negative, and PR negative postmenopausal patients received adjuvant treatment: six cycles of chemotherapy. The node- positive, ER positive, and PR positive postmenopausal patients received tamoxifen for two years. Node-negative patients received no adjuvant treatment. The median duration follow-up of living patients was 6.4 years. The number of deaths was 83, and the number of relapses was 108.

The biopsies were obtained with the agreement of the Institutional Review Board. Both Estrogen Receptor (ER) and Progesterone Receptor (PR) were determined by the dextran-coated charcoal method, as described previously [23]. The Laboratoire d'Oncologie Moleculaire Humaine is affiliated with the European Organisation for Research and Treatment of Cancer Receptor Study Group, which undertook the quality control of the assays (Koenders T, et al. 1983).

Table 1 : Patients and tumor characteristics (n=291)

Characteristics No of cases

Age <50 years 69 >50 years 222

Node involvement negative 134 positive 155 unknown 2

HPG

I 42

Il 1 1 1 III 109 unkown 29

Tumor type Ductular 212 Lobular 22 Others 57

Tumor diameter

< 2cm 21 2 to 5cm 188 > 5cm 68 unknown 14

ER

< 10 fmol/mg protein 88 > 10 fmol/mg protein 201 unknown 2

PgR

<10 fmol/mg protein 88

> 10 fmol/mg protein 200 unknown 3 PTP mRNA expression: Total RNA was isolated (RNeasy Mini Kit, Qiagen, France) from 40 mg of each tumour sample. Disruption and homogenisation of the tumour samples were performed using a Rotor-Stator Homogeniser (Ribolyzer, Hybaid). The amount of extracted RNA was quantified by measuring the absorbance at 260 nm. The quality of the RNA was checked by the ratio between the absorbance values at 260 nm and 280 nm, and then confirmed by electrophoresis of the RNA on a 1 .5% agarose gel containing ethidium bromide. Total RNA (2 μg) was reverse transcribed into cDNA using 5μM of random hexamers (Roche) and Superscript Il RNAse H- reverse transcriptase according to the manufacturer instructions (Invitrogen).

The real time Polymerase-Chain-Reaction (PCR) quantification of each gene was carried out on cDNA corresponding to 12.5 ng of total RNA using a Light Cycler 3 (Roche) with the LightCycler FastStart DNA MasterPLUS SYBR Green I Kit (Roche) according to the manufacturer instructions. Primer sequences and gene position, product length, time and temperature used for each gene are indicated in Table 2.

Table 2: PCR conditions and primers

To confirm the total gene specificity of the sequences chosen for the primers, we performed nucleotide-nucleotide BLASTN against a database of expressed sequence tags and nr (the nonredundant set of the GenBank, EMBL, and DDBJ database sequences). To avoid amplification of contaminating genomic DNA, one of the two primers was placed at the junction between two exons.

The relative quantification of PTP gene expression was performed with the comparative cycle threshold (CT) method where the CT parameter is defined as the cycle number at which the fluorescent signal is first detectable. This method is based on the use of a calibrator sample and an endogenous RNA control, which permits the quantification in the unknown samples. The human breast cancer cell line MCF7, known to express the three PTPs, was chosen as the calibrator sample (PTP expression = 1 ), and the hypoxanthine phosphohbosyltransferase 1 mRNA as the constant endogenous RNA. The relative PTP expression was given by the formula: 2- CT, where

CT = CT patient sample - CT calibrator sample; with CT = CT PTP - CT HPRT.

Statistical analyses: All the statistical analyses were done using the SPSS software (version 1 1 .0). Correlations between parameters were assessed according to the Spearman nonparametric test. Overall survival and relapse-free survival were studied by Kaplan Meier method analysis.

Comparison between curves was carried out by the log rank test. The proportional hazard regression method of Cox was used to assess the prognostic significance of parameters taken in association.

Results : T raining set: The classical correlations observed in breast cancer were found between ER and PR (p<0.001 , r = 0.761 ), the histoprognostic grade and ER (p = 0.05, r = - 0.256) or PR (p = 0.045, r = - 0.274), and tumour size and ER (p = 0.05, r = - 0.271 ). PTPL1 expression, positively correlated with the hormonal receptor status (p=0.02, r=0.307 between PTPL1 and PR).

When looking for the prognostic value the overall survival was significantly longer among patients with a high level of PTPL1 expression than among patients whose tumours had a low level of this protein (P=0.04 by the log rank test).

Testing set: The PTPL1 expression level, selected during the training set, was then determined in a complementary cohort of 232 breast cancer patients. In the total cohort (291 patients), the classical correlations observed in breast cancer were found between ER and PR (p<0.001 , r=0.626), ER and age (p<0.001 , r=0.268), and PR and age (p=0.01 , r=0.141 ). Hormone receptors were negatively correlated with histoprognostic grade (p<0.001 , r=- 0.366 and r=-0.342 for ER and PR respectively) and tumour size (p<0.001 , r=-0.229 and p=0.037, r=-0.126 for ER and PR respectively). Node involvement was positively correlated with tumour size (p=0.0015, r=0.19). PTPL1 expression was positively correlated with the hormonal receptor status and negatively with the histoprognostic grade and node invasion.

When looking for the prognostic value of PTPL1 , the overall survival was found to be significantly longer among patients with a high level of PTPL1 expression than among patients whose tumours had a low level of this protein (P=O.01 by the log rank test) (Figure 1 ). In Cox univariate analyses, in addition to PTPL1 expression, as expected, ER and PR expression, tumour size and node invasion were prognostic factors for global survival (Table 3). Table 3: Prognostic factors in Cox univariate and multivariate analyses

In multivariate analyses, PTPL1 was an independent prognostic factor for overall survival. PR and tumour size preserved their prognostic values and node invasion was no longer a prognostic factor for overall survival (Table 3). In the meantime, only node involvement and tumour size were prognostic factors for relapse free survival in univariate and multivariate analyses.

If we consider the N+ patients (n=155), relapse free and overall survival were significantly longer among patients with high level of PTPL1 expression than among patients whose tumours had low levels of this protein (respectively P=0.015 and p=0.043 by the log rank test) (Figure 2). Accordingly, in Cox univariate analyses PTPL1 was a prognostic factor for relapse free and global survival (P= 0.046, risk ratio = 0.55 and P=0.018, risk ratio = 0.40 respectively), but it did not preserve its prognostic value in multivariate analyses. Interestingly, it was observed that oestrogen receptor (ER)-positive patients (n=201 ) present an overall survival significantly longer among patients with high levels of PLPL1 expression (P=O.034 by the log rank test for ER+, p=0.3 for ER-) (Figure 3) compared to (ER)-positive patients expressing low level of PTPL1 . These results clearly indicate that PTPL1 retains its prognostic value for overall survival in patients with ER-positive tumours in spite the strong correlation of its expression with that of ER. The presence of oestrogen receptor/progesterone receptor is typically used as a rational basis for hormonal treatment. In view of these results, it can be conclude that PTPL1 could provide an additional criterion for implementation of such therapies, by better estimation of the overall survival prognostic of the treated patient.

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Claims

1 . A method for predicting the survival of a breast cancer patient consisting of determining the expression level of the PTPL1 gene in a biological sample obtained from said patient.

2. The method according to claim 1 wherein said biological sample is a cell or tissue sample.

3. The method according to claim 2 wherein said biological sample is a breast biopsy.

4. The method according to any of claims 1 to 3 wherein said expression level of the PTPL1 gene is determined by measuring the quantity of the PTPL1 mRNA in said biological sample obtained from said patient.

5. The method according claim 4, wherein said PTPL1 mRNA is selected in the group comprising SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO:4.

6. The method according to any of claims 1 to 3 said expression level of the PTPL1 gene is determined by measuring the quantity of the PTPL1 protein in said biological sample obtained from said patient.

7. The method according claim 6, wherein said PTPL1 protein is selected in the group comprising SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:8.

8. The method according to any of the previous claims, wherein the breast cancer patient is an oestrogen receptor (ER)-positive breast cancer patient.

9. The method according to any of claims 1 to 8, said method being also a method for typing the breast cancer and comprises the steps of: a) analysing a breast cancer marker chosen in the group comprising oestrogen and progesterone receptor expression, erb-2/HER2 expression, Ki-67, cyclin E, cyclin D1 , uPA, cathepsin D, or analysing a parameter selected in the group comprising regional lymph nodes invasion analysis, histological grade analysis, and the size of the tumour , and b) typing said breast cancer.