WO2005095980A1

WO2005095980A1 - Death-associated protein kinase (dap-kinase) as a new marker for breast cancer prognosis

Info

Publication number: WO2005095980A1
Application number: PCT/IB2005/001167
Authority: WO
Inventors: Anne Gompel; William Rostene
Original assignee: Institut National De La Sante Et De La Recherche Medicale (Inserm)
Priority date: 2004-03-31
Filing date: 2005-03-31
Publication date: 2005-10-13

Abstract

The present invention relates to a method for prognosis breast cancer comprising detecting DAP-kinase level of expression in breast cancer cells and determining the percentage of cells expressing DAP-kinase or the change of DAP-kinase expression level compared to a control sample as a predictor of the outcome and survival.

Description

Death-associated Protein Kinase (DAP-kinase) as a new marker for breast cancer prognosis

Background of the invention

Breast cancer is one of the most common causes of cancer-related deaths in women. Despite improvements in diagnosis and treatment of this disease in the past decades, the survival rates remain low in comparison with other cancers [1]. With advances in detection means of breast cancer, more patients are now diagnosed at earlier stages of the disease. However, it currently remains difficult to foresee whether those patients ^• will be cured with surgery alone or should benefit from additional and more aggressive treatments in order to improve their long-term survival.

It is therefore important that new, clinically relevant, and easily measurable markers be validated in breast tumors with the aim of improving initial staging and allowing a patient-to-patient therapeutic management. Patients with initial higher risk of recurrence or metastasis could benefit from appropriate adjuvant therapeutics in addition to complete surgical resection of the primary tumor.

Inactivation of tumor-suppressor genes and decreased cell apoptosis are essential mechanisms for breast tumorigenesis, contributing to deregulated tumor cell proliferation, invasion, and metastasis [2]. Tumor sensitivity to any given therapeutic regimen is commonly mediated by initiation of programmed cell death via available active .apoptotic pathways [3]. Several genes belonging to a defined apoptotic cascades, such as p53, have already demonstrated their association with breast tumor prognosis [4]. Death-associated protein (DAP) kinase is a novel multidomain calcium/calmodulin-regulated and cytoskeletal associated serine/threonine kinase mandatory for IFN-γ (interferon gamma), TNF-c (tumor necrosis factor alpha) and activated Fas-induced apoptotic cell death and detachment from the extracellular matrix, comprising modules such as ankyrin repeats mediating protein-to-protein interactions as well as a death domain [5]. This 160 Kd protein kinase is normally localized in the cytoskeleton in association with actin microfilaments. The death promoting effects of DAP-kinase depend on its intact catalytic activity, the correct intracellular localization, and the presence of the death domain [6]. It was found that DAP-kinase mRNA and protein expression is lost in cancer cell lines including B-cell lymphoma and leukemia cell lines [7, 8]. DAP-kinase expression was also shown to correlate strongly with recurrence and metastasis incidence in small cell lung cancer, B-cell malignancies, primary head and neck tumors, colon and bladder cancers, and multiple myeloma [6, 9]. Restoration of DAP-kinase expression to physiological levels in a murine model of highly metastatic lung carcinoma cells can strongly suppress their metastatic ability [10]. A. fraction of lobular invasive breast carcinoma exhibited a high level of DAP-kinase loss of expression (mRNA) and promoter hypermethylation [16]. DAP kinase inactivation was also correlated with estrogen receptor positivity and the absence of p53 expression. However, no correlation was found between DAP kinase promoter methylation and DAP-kinase protein level, tumor size, grade or node involvement, and patient survival was not studied [16]. Thus, there is no data connecting DAP-kinase protein expression and breast cancer.

In the context of the invention, we have analyzed the level of DAP-kinase expression in breast cancer specimens and its correlation with survival among the 128 patients. 30 patients showed a DAP-kinase staining <20% while 98 had a staining over 20%. Mean follow-up time was 62 months. The association between tumor SBR grade (p=0.009), ER and PR expression (p=0.002 and 0.001, respectively), tumor size (p=0.05), Bcl-2 expression (p=0.004) and DAP-kinase immunostaining in the ductal carcinoma group was highly significant. We observed a significantly longer survival (64 months) in the high DAP-kinase expression group compared with the women whose tumors showed a loss of DAP- kinase expression (51 months). DAP-kinase protein was strongly expressed in normal breast tissue and in human breast epithelial cells primary cultures. Estradiol decreased DAP-kinase expression in these cells, arguing for hormonal regulation of the protein.

Loss of DAP-kinase expression negatively correlates to survival and positively correlates to the probability of recurrence in a very significant manner. Therefore, DAP-kinase constitutes a novel and independent prognosis marker for breast cancer.

Description

Therefore, in a first embodiment, the invention is aimed at a method for prognosis breast cancer comprising detecting DAP-kinase level of expression in breast cancer cells and determining the percentage of cells expressing DAP-kinase, a percentage < 35%, 30%, 25% or 20% being indicative of a poor outcome. Alternatively, a percentage of cells expressing DAP-kinase above 35%, 30%, 25% or 20% is indicative of a favorable prognosis.

The expression "poor outcome" is meant to refer herein to a shorter overall mean survival rate compared to the overall breast cancer population. For example, for a DAP-kinase expression rate below 20%, the survival rate is currently of about 43 months, but it may change with new therapeutics being developed. On the other hand, for a DAP-kinase expression rate above 20%, the survival rate is currently of about 63 months. Also and as explained hereinafter, the DAP-kinase expression rate in breast cancer cells below 20% (poor outcome) is correlated with SBR grade 3 tumors and other makers of tumor aggressiveness, such as tumor size, ER expression and Bcl-2 expression. The SBR Grade stands for Scarff Bloom Richardson Grade. This grade is a way of determining how much differentiated a tumor is. Invasive cancers can be Grade I, which is the best differentiated. Grade III is the less differentiated and is the most aggressive type of tumor and Grade II is in the middle. In general, the prognosis is best for Grade I tumors, poorest for Grade III tumors and somewhere in the middle for Grade II tumors.

The presence of estrogen and progesterone receptors is associated with a more favorable prognosis and a greater likelihood that the tumor will respond to hormonal therapy such as Tamoxifen. Here, a DAP-kinase expression rate below 20% was also found to correlate with the loss of expression of these receptors.

The detection of DAP-K expression in breast cancer cells can be performed with any methods currently employed by the man skilled in the art.

For example, one can detect DAP-K expression in cells by immunohistochemistry. It basically consists of staining tissue section with the antibodies against DAP-kinase. The antibodies according to the invention are, for example, monoclonal or polyclonal antibodies or Fab or F(ab')2 fragments thereof. They may also be in the form of immunoconjugates or of labelled antibodies (immunofiuorescence, gold labelling, enzymatic immunoconjugates) so as to obtain a detectable and/or quantifiable signal (Harlow, E., and D. Lane. 1988. Antibodies A Laboratory Manual. Cold Spring Harbor Laboratory, pp. 53-242). Alternatively, if a non labelled mouse DAP-kinase antibody is used, the method further comprises a step consisting of incubating with an antimouse immunoglobulin coupled with a label (fluorescent or enzymatic for example). For example, it is possible to use a biotinyl goat antimouse immunoglobulin and the detection is performed by incubating with the streptavidin biotin peroxydase complex and its substrate.

Thus, the method of the invention can include the steps of: a) Rinsing slides in TBS (for few minutes) b) Removing excess liquid from around specimen c) Applying normal serum to cover specimen and incubating d) Tapping off serum e) Applying appropriate quantity of enzyme-conjugated primary anti-DAP-K antibody and incubating f) Applying substrate-chromogen solution and incubating until the desired color intensity has developed.

Alternatively, step e) may consist of applying primary anti-DAP-K antibody and incubating, repeat step a) and b); followed by step f) which is applying enzyme- conjugated secondary antibody directed against primary antibody immunoglobulin and g) applying substrate-chromogen solution and incubating until the desired color intensity has developed.

Thus, the invention is directed to the method as defined above comprising contacting breast cells with a DAP-K antibody, directly or indirectly labelled, detecting the signal and determining the ratio of cells expressing DAP-K.

The invention also contemplates the above method wherein the expression of DAP-K in a given tissue is globally evaluated by combining quantitative RT-PCR and linear discriminant analysis.

It is also possible to use cDNA micro-array technology (M. Schena, D. Shalon, R. W. Davis, and P. O. Brown, "Quantitative monitoring of gene expression patterns with a complementary DNA microarray," Science, 270(5235), 467-70, 1995). The method here is to quantitatively analyze fluorescence signals that represent the relative abundance of mRNA coding for DAP-K from two distinct tissue samples. Two different samples of mRNA (one normal sample control and one from the patient can be labelled with different fluorescent molecules and then co-hybridized on to arrayed DAP-K gene. Ratios of gene-expression levels between the samples are calculated and used to detect meaningfully different expression levels between the samples (US 6,245,517). Other examples include high density tissue microarray technology involving arraying up to thousands of cylindrical tissue cores from individual tumors on a tissue microarray (Kononen et al. Nat Med. 1998 July ;4(7):844-7). This technology allows rapid analysis of a large number of samples so that the statistical relevance is determined in a single experiment. Arrays have been made containing different tumor types (Schraml et al. Clin Cancer Res. 1999 August ;5(8): 1966-75) and multiple stages and grades within one tumor type (Bubendorf et al. Cancer Res. Feb. 15, 1999 (4):803-6 and Bubendorf et al. J Natl Cancer Inst. Oct. 20, 1999 ;91(20): 1758-64). This technology is now considered useful for rapidly characterizing the prevalence and prognostic significance of differentially expressed genes identified using cDNA array technology. Tissue microarrays have also been useful to study the expression patterns of putative tumor suppressor genes (Bowen et al. Cancer Res. Nov. l, 2000;60(21):6111-5).

It is also possible to use gel electrophoresis for detecting DAP-K in a sample. For example, the invention encompasses a method for detecting DAP-K level in a breast tissue sample comprising 2D-gel electrophoresis and Mass Specfrometry, in particular Surface-enhanced laser desorption and ionisation time of flight (SELDI-TOF) Mass Specfrometry. Here, the purpose is to obtain proteomic profiling of normal sample versus DAP-K positive breast cancer sample so as to directly detect the level of DAP- K expression with such profiles. In this regard, mass spectrum are obtained from test samples, which generate signature patterns (plot relative abundance of key discriminatory proteins including DAP-K). General process for pattern discovery and pattern matching are described in Petricoin F, Use of proteomic patterns in serum to identify ovarian cancer, The Lancet, Vol. 359, Feb 16, 2002 ; Zhao Rui et al, Use of serological proteomic methods to find biomarkers associated with breast cancer, Proteomics, Vol. 3, Issue 4 , p 433-439, 2003. A diagram representing this method is illustrated at fig 1 of Sandy Kennedy, Toxicology Letters 120(2001) 379-384, incorporated herein in the description. It is further envisioned to profile fluids proteins from breast cancer DAP-K+, DAP-K- patients and also control sample to identify surrogate fluids makers.

In a second embodiment, the invention is directed to a kit for performing the method as defined above. This kit can comprise either a labelled DAP-K antibody or a first DAP- K antibody and a second labelled antibody directed against said first antibody. In- another aspect, the kit can comprise the primers for specifically amplifying DAP-K mRNA or cDNA, such as primers for performing q-RT-PCR for example and/or a DAP-K c-DNA array. In this regard, specific primers and probes can be designed for example by referring to the sequence available in NCBI under the accession number BC002726 (gi:33877086). The primers herein are selected to be "substantially" complementary to the above DNA sequence. This means that the primers must be sufficiently complementary to hybridize under stringent conditions with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment can be added to the 5' end, with the remainder of the primer sequence being complementary to the strand. Also, longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementary with the sequence to hybridize therewith and form the template for synthesis of the extension product. One type of quantitative PCR assay involves simultaneously amplifying control DNA which amount is known and samples suspected to contain a target sequence. Following amplification, the amounts of amplified products (amplicons) are compared (Clementi el al., Competitive polymerase chain reaction and analysis of viral activity at the molecular level. GATA 1994; 11:1-6 and Kahn et al., Neurosci. Lett. 1992; 147:114- 117). In other methods, the control molecule is similar to the target DAP-K mRNA (quantitative-competitive PCR (QC PCR)). Following competitive amplification, the two products synthesized (amplicons) are distinguished, for example, by size using gel electrophoresis (Becker-Andre, Quantitative Evaluation of mRNA levels. Meth. Molec. Cell Biol. 1991 2:189-201.) A more recently developed type of quantitative PCR assay is the 5'-nuclease assay and "real-time PCR." (Gibson et al., A novel method for real time quantitative RT-PCR Genome research. 1996; 6:995-1001; Heid et al., Real-time quantitative PCR. Genome Research. 1996; 6:986-994; and Livak et al., US 5,538,848). This method is based on probes that are DNA sequences labeled with two different fluorescent dyes, for example, a reporter dye and a quenching dye. Kits from the Applied Biosystems are available under the trademark TaqMan™ and fluorescence can be monitored throughout the PCR amplification with the Applied Biosystems ABI PRISM 7700 for example.

Thus, the kit of the invention may comprise pre-labelled primers and optionally reagents as described in the above documents for q-PCR, QC-PCR and real-time PCR.

In a third embodiment, the invention is directed the use of the method and kit defined above for the prognosis of patients afflicted with breast cancer. It also relates to the use of the method and kit for the initiation of adequate therapy early in the course of the disease, for providing an ex vivo assessment of the antitumor effects of the chemotherapy in the course of the therapy.

Thus, the invention is aimed at the use of a method or kit as defined above as a predictor of breast cancer prognosis and survival.

Indeed, this is the first time that DAP-kinase level of protein expression is correlated to breast cancer prognosis and survival.

In the present invention, the loss of DAP-kinase protein expression very strongly correlated with disease recurrence and disease-free survival duration. At 5 years of follow-up, the overall survival rate was significantly higher in the group expressing high DAP-kinase levels compared with the low expression group, 0.88 and 0.57 respectively (see Figure 2).

This observation remained valid when adjusted for other prognosis markers (tumor size, ER expression, SBR grade, and Bcl-2 expression). The 20% cut-off level of DAP-kinase staining was the first one to demonstrate a significantly different clinical outcome. When groups were split with 25, 30 and 35% invasive cell staining, the difference remained significant. The decrease in DAP-kinase protein expression observed in cultured normal breast cells seems to be the consequence of a direct E2 effect, as suggested by antiestrogen reversion. This demonstrates that DAP-kinase is yet another target for E2 in apoptosis regulation. We recently reported that E2 was able to alter the cellular expression levels of Bcl-2, p53 and caspase-3 in HBE cells; this effect being also reversed by an antiestrogen agent [15]. However, stratifying the data by anti-estrogen adjuvant treatment did not significantly modify the prognosis association to DAP-kinase level of expression. This result warrants confirmation since the group of patients having received Tamoxifen and exhibiting low levels of DAP-kinase expression was very small (n=7). In addition, E2 is probably not responsible alone for DAP-kinase regulation, since only the ERα-negative cells (MDA-MB361) expressed DAP-kinase, whereas the ERα-expressing cell lines remained negative as previously reported [8]. The loss of expression of DAP-kinase in the 3 cell lines may be related to their specific tumoral phenotype. A recently published study showed that the DAP-kinase promoter was highly methylated in MCF-7 cells, while methylation was absent in MDA-MB231 cells. The degree of promoter methylation, as an epigenetic regulation, could be partly responsible for the variable level of DAP-kinase protein expression in breast tissue. This phenomenon seems to be a gene and histological type-specific one [16].

A very low level of promoter methylation was reported in normal breast tissue. Only 9% of their 85 invasive ductal carcinoma specimens showed promoter hypermethylation. In our series, loss of DAP-kinase expression clearly predominated in invasive ductal specimens, while only 2 of the 23 lobular tumors exhibited low DAP-kinase expression (less than 20% of stained cells). It is thus likely that promoter epigenetic hypermethylation is only one possible way of inactivating a gene at the protein level as suggested from the RNA analysis. Indeed, significant part of this sample did not express DAP-kinase without methylation. Others have reported promoter homozygous deletions of CpG island inducing DAP-kinase loss of expression in invasive pituitary tumors [17]. Very little is known about the mechanisms by which DAP-kinase achieves its pro- apoptotic function. A recent study demonstrated that DAP-kinase activates a p53- mediated pathway [18]. DAP-kinase was also described as a negative regulator of integrin activity and cell adhesion, diminishing integrin-mediated survival signals, and that integrin activation blocked DAP-kinase-induced upregulation of p53 [19]. Thus, the DAP-kinase pro-apoptotic action seems to locate early in the p53-dependent apoptotic pathway; thereby explaining the fact that DAP-kinase inactivation and p53 overexpression are mutually exclusive [17].

In a recent study, a shorter 5-year survival rate was observed in patients with non-small cell lung cancer with DAP-kinase promoter CpG region hypermethylation [20]. DAP- kinase was also shown to be commonly inactivated by promoter hypermethylation in B-cell malignancies [8, 21] and multiple myeloma (MM) [9]. High fatality and poor treatment outcomes in MM are linked to dissemination forms and uncontrolled circulatory pool of MM precursors. It is possible that the cellular selective advantage conferred by DAP-kinase inactivation may play a role in the induction and maintenance of circulatory tumor pool.

Whether poor prognosis for patients expressing low levels of DAP-kinase protein is directly related to local enhanced tumor cell survival, invasiveness potential, and metastasis proneness remains to be established. Whether DAP-kinase loss of expression can also be observed in a subset of very, early stage breast tumors or in certain types of benign atypical hyperplasia may be important for appropriate management of benign breast disease and in situ breast cancer.

In conclusion, in a series of 128 patients followed-up for sixty months, we show that DAP-kinase protein expression constitutes a new and strong independent predictor of breast cancer prognosis and survival.

The invention is further described in the examples and figures below.

LEGENDS Figure 1: Immunohistochemical staining for DAP-kinase on breast tumor sections, using an anti-DAP-kinase monoclonal antibody.

(A) Normal breast tissue and intra-ductal carcinoma expressing a high level of DAP-K (more than 80% of the cells are stained), magnification x20. (B) Intraductal carcinoma showing heterogeneous staining for DAP-K, x40.

(C) Lobular carcinoma, 80% to 100% of the cells are stained, x20. D) Invasive ductal carcinoma showing a significant loss of DAP-K expression, x20.

Figure 2: Kaplan-Maier analysis for disease-free and global survival duration in both low (<20%) and high (>20%) DAP-kinase expression groups of the 105 patients with invasive ductal breast carcinoma.

(A) Probability of disease-free survival for patients with high DAP-kinase expression (n=77) versus patients with low DAP-kinase expression (n=28).

(B) Probability of overall survival for patients with high DAP-kinase expression (n=77) versus patients with low DAP-kinase expression (n=28).

Figure 3: DAP-kinase protein levels in HBE cells under E2 and antiestrogen treatments.

HBE cells were treated for 48h with lOnM E2 alone or with lμM RU 58668. DAP- kinase protein content was estimated by western blot. The relative amounts of proteins are quantified by scanning densitometry using the software program RAG (Biocom, France). A representative Western blot is shown above the graph. Data are expressed as percent compared to control. Values are expressed as the mean ± SEM of four separate experiments. * p<0.05 compared to control and E2+RU

EXAMPLE 1: Correlation between DAP-K expression level, overall survival rate and other tumor aggressiveness markers.

1.1 Patients and Methods 1.1.1 Study population.

A total of 128 patients who had been diagnosed with breast cancer and undergone tumorectomy or mastectomy for complete resection of their primary tumors at the Hotel Dieu Hospital of Paris Gynecology Department, during the period from June 1985 through May 1998 were included in the study. Patients eventually received adjunct chemotherapy and/or radiotherapy according to initial staging. Fifty seven postmenopausal patients with invasive ductal carcinoma staining positively for estrogen receptor (ER) received postoperative hormone therapy with Tamoxifen for a mean period of 5 years. Patients were followed-up by the same physician for a mean follow-up time of 66 months. Survival and follow-up durations were measured as the time between the first oncology consult after treatment completion and the last consult in the department, or death. Patient records were reviewed retrospectively for demographical characteristics, clinical data, outcome and survival. Tissue sections were obtained from each tissue block for immunohistochemistry staining, while precise histological diagnosis was confirmed for each case by the same pathologist. ImmunoMstochemistry. The 128 breast tumors were analyzed by immunohistochemistry for DAP-kinase, ER, PR and Bcl-2 staining, using an anti- DAP -kinase monoclonal antibody as previously described [5, 7], an anti-ER-c monoclonal antibody (TEBU, Santa Cruz Biotechnology, mouse monoclonal antibody, Le Perray en Yvelines, France), an anti-PR monoclonal antibody (TEBU, Santa Cruz Biotechnology, mouse monoclonal antibody, Le Perray en Yvelines, France), and an anti-Bcl-2 monoclonal antibody (Dako Cytomation S.A., Trappes, France) on paraffin tumor blocks. Briefly, 8 μm paraffin-fixed tissue sections were deparaffined and rehydrated. To permeate the cells, slides were immersed in citrate buffer 10 mM (pH^ό) and micro waved 3 times for 5 minutes. After rinsing thoroughly with TBS (Tris Buffer Salin, containing casein and tween), cells were stained with the antibodies against DAP-kinase, ER- , PR and Bcl-2 (dilution 1/200, 1/100, 1/50, and 1/50, respectively) for 2 hours at room temperature. Slides were washed 3 times with TBS for 5 minutes, then incubated with the biotinyl goat antimouse immunoglobulin coupled to biotin (dilution 1/200) for 20 minutes (Nalbiotech, France), followed by 3 TBS washes. The streptavidin biotin peroxydase complex was applied in TBS (dilution 1/200) to the sections for 45 minutes, followed by 3 TBS washes. Slides were flooded with 8 mg-DAB (diaminobenzidine) hydrogen peroxide chromogen in the dark for 5 minutes, and further rinsed with distilled water. Finally, slides were immersed in hematin for 30 seconds, and mounted for light microscopy analysis. The resultant staining was evaluated both for DAP-kinase and Bcl-2 by determination of the percentage of stained invasive tumor cells on a given paraffin slide. Staining for DAP- kinase and Bcl-2 were achieved on two serial sections belonging to the same paraffin block.

1.1.2 Human breast epithelial cells (HBE) cultures.

Breast tissue was obtained from 5 women in ages between 15 to 25 years undergoing reduction mammoplasty. The patients had no history of breast disease and pathological analysis showed only normal breast tissue.. Sampling of the tissue was performed according to the French governmental regulations on clinical experimentation. The epithelial cells were plated and grown as previously described [11]. Briefly, the tissue was digested with 0.15% collagenase (Roche Diagnostics, Meylan, France) and 0.05% hyaluronidase (Sigma- Aldrich Chimie, Saint Quentin Fallavier, France) in Ham's F10 medium (Invitrogen, USA), and filtered through 150-μm sieves to retain undigested tissue. Cells were grown in Ham's F10 medium with phenol red, supplemented with 0.24% NaHCO3 (Invitrogen), 1% penicillin (10000U) / streptomycin (10 mg) (Sigma), 5 ng/ml cortisol (Sigma), 6.5 ng/ml triodothyronine (Sigma), 10 ng/ml choleratoxin (Sigma), 5 mg/ml transferrine (Sigma), 5% compatible human serum, 0.12 U/ml insulin (Sigma), and 10 ng/ml epidermal growth factor (Sigma), in a humidified atmosphere of 5% CO2, 95% air. For comparison in the level of DAP-K expression, breast cancer cell lines were also grown. MCF-7, T47-D and ZR75-1 cell lines were a gift from Dr C Mercier-Bodard (Kremlin-Bicetre, France) and originally came from the laboratories of Dr M Lippman (Bethesda, USA) and Dr K Horwitz (Denver, USA). MCF-7, ZR75-1 and MDA-MB231 cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, France) without phenol-red, supplemented with 5% fetal calf serum and 2mM glutamine, T-47-D in Roswell Park Memorial Institute 1640 (RPMI) supplemented with 5% fetal calf serum and 2mM glutamine (Invitrogen, France).

1.1.3 Hormonal treatments. Before hormonal treatments, HBE cells were synchronized in a medium containing 20 μM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, for 40h in Ham F10 w/o phenol red, and synchronization was stopped by adding 2mM mevalonate to the hormone-containing medium [12]. Subsequently, cells were treated 48h in a phenol red free medium containing 5% of compatible human serum with lOnM estradiol (E2) and 1 μM of a potent steroidal antiestrogen RU58668 (RU58).

1.1.4 Western Immunoblotting.

DAP-kinase protein expression was measured in cultured normal human epithelial breast cells by Western blot technique as previously described [13] and modified as following. Extraction of the proteins was performed using a buffer containing DOC 0.5%, SDS 0.1%, NP40 1% and 2 antiproteases, PMSF, 100 μg/ml (Boehringer) and aprotinin (Sigma- Aldrich), 1 μg ml. The extracted proteins were measured using BCA (Pierce, Interchim). Eighty micrograms of protein from HBE cells were analyzed by 8% Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis. DAP-kinase immunoreactivity was detected using a mouse monoclonal antibody at 1:2500 provided by A.Kimchi [5, 7]. Relative amounts of proteins were quantified by scanning densitometry using the software program RAG (Biocom, France).

1.1.5 Statistics. Association between the level of DAP-kinase expression and the various prognosis markers was analyzed with SPSS and BMDP softwares using chi-square and Student t- tests. Correlation with disease-free survival time was performed by Kaplan-Maier and Cox model survival analysis. Nonparametric Mann Whitney tests were used to compare the effects of hormonal treatments in HBE cells. One-way ANONA test and multiple range Student-Νewman-Keuls tests were performed to compare the relative efficiency of each treatment as was previously described [2]. 1.2 Results

A total of 105 invasive breast ductal carcinoma and 23 lobular carcinoma specimens were included. Patients' mean age was 55.8 ± 1.3 years (range 29 to 89 years) at the time of diagnosis. Twenty patients died during follow-up (mean disease-free survival time, 59 months), while 108 patients were still alive after a mean follow-up time of 66 months (mean disease-free survival time, 64 months). The mean overall survival time was 62 months. Among the 20 patients who died, 15 died from breast cancer, 3 of heart disease and 2 of unknown cause. The general clinical characteristics of the patients, stratified by histological type, are shown in Table 1.

Table 1: Patient clinical characteristics stratified by tumor histological type.

Histological Type [n patients (%)] Ductal Lobular n=105 (82%) n=23 (18%) P *

Age in years [mean (SE)] 56.3 ± 1.3 53.5 ± 2.3 NS*

BMI [mean (SE)] 23.9 ± 0.4 23.4 ± 0.7 NS

Menopausal status Patient No [n (% of patients)] 65 (62%) 14 (61%) Age at menopause in years [mean (SE)] 52.0 ± 0.3 51 ± 0.7 NS

HRT* [ever users] Patient, No [n (% of patients)] 17 (26%) 8 (57%) NS HRT, Duration in months [mean (SE)] 58.4± 15.2 46.5 ± 14.1 NS

Parity No of children [mean (SE)] 1.6 ± 0.2 1.8 ± 0.3 NS

Breast feeding Patients No [n (% of patients)] 36/77 (47%) 10/20 (50%) NS Cumulative duration in months [mean 6.3 ± 2.1 4.2 ± 0.9 NS (SE)]

Benign breast disease * Patients No [n (% of patients)] 21 (20%) 5 (22%) NS

Current smoking [n (% of patients)] 22/85 (26%) 8/23 (35%) NS

Tumor size (cm) [mean +/- SE] 2.0 ± 0.1 1.5 ± 0.2 0.07

Invaded nodes [n, number of cases studied] 38/101 10/21 (48%) NS ^' (18%) Disease-specific deaths during follow-up 17 1 0.13 5-years survival probability (95% CI) Overall (deaths) 17 (17%) 3 (13%) NS Disease-specific (recurrence) 17 (17%) 1 (4%) 0.13 Disease-free survival in months 57.4 ± 3.7 65.9 ± 9.0 NS

* Statistical significance is defined as a p value < 0.05; NS, non significant

* HRT, hormone replacement therapy

* Benign Breast Disease is considered as present when breast examination, mammogram, and breast sonogram coincide with the diagnosis

Tumor size, node invasion status, histological Scarff-Bloom and Richardson (SBR) grading, estrogen and progesterone receptor expression, and Bcl-2 expression data in the 105 invasive ductal carcinoma specimens are presented in Table 2. Among the 105 patients, 28 showed a DAP-kinase staining < 20%, 77 had a staining over 20%, while more than 80% of adjacent normal breast cells showed intense DAP-kinase staining in all the cases where normal cells were present and staining could be quantified (see Figure la to Id). DAP-kinase loss of expression was mainly observed in SBR grade 3 and ER negative tumors, while grade 1 and ER positive tumors displayed high expression levels. More specifically, tumor SBR grade (p=0.009), ER (p=0.002) and PR (p=0.001) expression, tumor size (p=0.05) and Bcl-2 (p=0.004) expression were significantly associated with the DAP-kinase immunostaining level (see Table 2).

Table 2: Outcome data and prognosis markers stratified by DAP-kinase expression level in the 105 invasive ductal carcinoma.

DAP-kinase positive invasive tumor cells [%] < 20% > 20% P * n=28 n=77 Age in years [mean (SE)] 55.4 ± 3.3 56.6 ± 1.4 NS Menopausal cases [n (% of patients)] 15/28 50/77 NS HRT [ever users, n patients / N cases studied] 2/27 15/74 NS Tumor size in cm [mean (SE)] 2.3 ± 0.3 1.8 ± 0.1 0.05 Invaded Nodes [n / N cases studied^' 12/26 26/75 NS Positive Estrogen Receptor [n patients / N cases studied] 14/28 61/73 0.002 Positive Progesterone Receptor [n patients (% of patients)] 13/28 62/72 0.001

SBR* Grade: 1 7 31 2 10 35 3 10 8 ^" 0.009 (n=101 studied cases) n=27 n=74

Tamoxifen use during follow-up 7/28 50/77 0.0006

Bcl-2 (% of marked invading tumor cells +/- SE) 43.9 ± 6.9 69.5 ± 4.4 0.004 (n=74 cases studied) [Mean +/- SE] n=19 n=55

Metastasis during follow-up [n / N cases studied] 5 6 0.14

Recurrence during follow-up [n patients/ N cases 11 8 0.004 studied]

Deaths during follow-up 10 8 0.006

Thus, DAP-kinase expression follows closely the other well recognized breast cancer prognosis markers. When Kaplan-Maier analysis was performed, stratified on histological type, overall (64 months) and disease-free (63 months) survival in the high DAP-kinase expression group was significantly longer compared with the women whose tumors showed a loss of DAP-kinase expression (51 and 43 months, respectively) (see Figure 2). The higher DAP kinase staining, the better was the overall survival prognosis (p<0.005). This result remained valid after adjusting for age, tumor size, estrogen receptor positivity, node involvement, in the 96 cases studied (data not shown).

Finally, using a Cox model analysis, DAP-kinase staining remained an independent prognosis marker after adjustment for SBR grade, ER and PR expression, tumor size , tamoxifen use and Bcl2 expression. Among the 57 Tamoxifen-treated patients with ductal carcinoma, 50 had a DAP-kinase staining over 20%, a disease-free and overall survival of 64 and 66 months respectively, 22% were node positive. The Tamoxifen treated patients (n=7) exhibiting a low DAP-kinase staining had a significantly lower disease-free (55 months) and overall (62 months) survival (p=0.006); and 43% were node positive. Thus, it appears that hormonal adjuvant therapy does not modify prognosis association to DAP-kinase level of expression. Since the DAP-kinase expression was consistently high in the normal breast tissue adjacent to the tumor cells, and since its expression was strongly correlated to ER positivity, we used primary cultures of normal human breast cells (HBE cells) developed routinely in our laboratory, to study estradiol (E2) regulation of DAP-kinase expression in normal,breast cells [2]. We have previously reported that the normal HBE cultures still express ER and PR under our experimental conditions, and thus remain responsive to hormones and anti-hormones for proliferation and apoptosis measures [11, 14]. As shown in Figure 3, and as expected, DAP-kinase appeared as a single band of 160kD in the HBE cell cultures. In contrast, DAP-kinase was not detected in the 3 studied hormone-dependent cell lines (MCF-7, T47-D and ZR75-1 cells). A very weak expression was detected in the ER-α negative cell line, MDA- MB231 (data not shown). In addition, the DAP-kinase protein level of expression was modulated by E2 in the HBE cells. A constant, moderate but significant decrease in DAP-kinase expression was observed under cellular E2 treatment (p<0.05) (see Figure 3). This effect was reversed by a potent antiestrogen arguing for an ER-mediated effect (E2+RU58, NS compared to control and p<0.05 compared with E2).

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Claims

1. A method for prognosis breast cancer comprising detecting DAP-kinase level of expression in breast cancer cells and determining the percentage of cells expressing DAP-kinase, a percentage <35%, more particularly 20%, being indicative of a poor outcome.

2. The method according to claim 1, wherein a percentage of cells expressing DAP- kinase above 20% or 35%, is indicative of a favorable prognosis.

3. The method according to claim 1, wherein a DAP-kinase expression rate below 20% indicate a survival rate of about 43 months.

4. The method according to claim 1, wherein a DAP-kinase expression rate above 20% indicate a survival rate of about 63 months.

5. The method according to claim 1, wherein a DAP-kinase expression rate below 20% correlates with SBR grade 3 tumors and other makers of tumor aggressiveness, such as tumor size, ER expression and. Bcl-2 expression.

6. The method according to one of claims 1 to 5, wherein a DAP-kinase expression rate is determined by immunohistochemistry.

7. The method according to claim 6 comprising staining directly or indirectly tissue section with an anti-DAP -kinase antibody.

8. The method according to claim 7, wherein said antibody is a monoclonal or polyclonal antibody or a Fab or a F(ab')2 fragment thereof.

9. The method according to claim 7, wherein it comprises the use of an anti-DAP- kinase primary antibody and a staining with a labeled-secondary antibody directed against said primary antibody immunoglobulin.

10. The method according to claim 7, wherein it comprises the use of a labelled anti- DAP -kinase primary antibody.

11. The method according to one of claim 9 and 10, wherein said labelled antibody is a fluorescent, gold or enzyme immunoconjugate.

12. The method according to one of claims 6 to 11 comprising detecting the signal and determining the ratio of cells expressing DAP-K.

13. A method for prognosis breast cancer comprising detecting DAP-kinase level of expression in breast cancer cells and determining the level of expression of DAP- kinase compared to the expression level in control sample, a significant decrease of the level of expression in breast cancer cells being indicative of a poor outcome.

14. The method according to claim 13, wherein the expression of DAP-K in a sample is evaluated by combining quantitative RT-PCR and linear discriminant analysis, competitive quantitative PCR, cDNA micro-array technology, or tissue microarray technology.

15. A kit for performing the method as defined in one of claims 1 to 12 comprising either a labelled DAP-K antibody or a first DAP-K antibody and a second labelled antibody directed against said first antibody.

16. A kit for performing the method as defined in one of claims 13 to 14 comprising the primers for specifically amplifying DAP-kinase mRNA or cDNA.

17. The use of a method defined in one of claims 1 to 14 and kit defined in one of claims 15 to 16 for the prognosis of patients afflicted with breast cancer.

18. The use according to claim 17 for the initiation of adequate therapy early in the course of the disease.

19. The use according to claim 17 for providing an ex vivo assessment of the antitumor effects of the chemotherapy in the course of the therapy.

20. The use according to claim 17 as a predictor of breast cancer prognosis and survival.