GB2379738A

GB2379738A - Diagnosis of presence of invasive tumour cells

Info

Publication number: GB2379738A
Application number: GB0122410A
Authority: GB
Inventors: Miriam Victorine Dwek; Anthony John Cullen Leathem
Original assignee: AGAINST BREAST CANCER
Current assignee: AGAINST BREAST CANCER
Priority date: 2001-09-17
Filing date: 2001-09-17
Publication date: 2003-03-19
Anticipated expiration: 2021-09-17
Also published as: GB2379738B; GB0122410D0

Abstract

Presence or absence of invasive phenotype tumour cells particularly in breast cancers is established by profiling the oligosaccharides present in tumour cell samples e.g. using HPLC or electrophoresis. The presence of "discontinuous higher molecular weight" oligosaccharides is indicative of invasive cell presence. Kits for the diagnosis are also disclosed and monoclonal antibodies specific for oligosaccharides or for a protein displaying oligosaccharides are also claimed.

Description

CANCER DIAGNOSIS The present invention relates to methods for establishing the likelihood of benign tumours metastasising, to kits for such methods and to treatment for such tumours.

Breast cancer is the most common female cancer, with the UK having among the highest incidence rate in the world. Over 30,000 patients are diagnosed each year in this country, 80% of these being post-menopausal women.

The functional unit of the breast is composed of ductules and lobules. The lobules are composed of acini which produce milk in the lactating female. The milk then passes into a series of ductules leading to larger lactiferous ducts and a lactiferous sinus, in which the milk is stored. The remainder of the breast acts to support the lobules and ducts and consists of dense fibrous connective tissue and adipose tissue. Breast cancers arise from the epithelial cells at the junction of the duct and the Terminal Ductule Lobular Unit (TDLU) and are broadly divided into either lobular or ductal types, and then further into in situ or invasive carcinoma. In situ carcinoma is contained within the basement membrane of the ducts or lobules. An invasive cancer arises when malignant cells invade into the adjacent tissue.

There are two types of in situ breast cancer, known as Ductal Carcinoma In Situ (DCIS) and Lobular Carcinoma In Situ (LCIS). DCIS arises within the ducts of the breast and is a complex pathological condition in which cancer cells proliferate within the duct yet do not invade the surrounding stroma. These types of breast cancer are frequently diagnosed by mammography, the x-ray technique used during breast screening for the identification of breast cancer. There are few treatment options for patients with DCIS, although it is recognised that ten years after diagnosis, approximately only 30% of patients with in situ disease develop invasive cancer.

Mastectomy is currently the preferred option for treatment. However, since two-thirds of cases of DCIS do not develop into invasive cancer, approximately 60% to 70% of mastectomies are carried out unnecessarily, as the in situ carcinoma could be removed safely without the need for a full mastectomy. Mastectomies are

expensive, and traumatic for the patient and, therefore, there is a need for a way of assessing whether an in situ carcinoma will remain as such, or whether it is likely to develop into an invasive carcinoma and, therefore, require a mastectomy.

Complex carbohydrate sugar residue multimers, or oligosaccharides, are found on the surface of mammalian cells forming the glycocalyx. These sugar residues are part of glycoproteins or glycolipids. The cell surface expression of oligosaccharides changes throughout development of the maturing organism, until specific expression is restricted to a specific cell type (Alberts et al., 1994, Molecular Biology of the Cell). Fukuda showed that this regimented expression was lost in carcinogenesis and that neoplastic cells altered their surface oligosaccharide expression patterns [Fukuda et al., Cancer Research 1996 (56); pp. 2237-2244].

Two changes in cell surface oligosaccharide expression thought to be . Lw AAariL 1 11 11 11 L & I Al LILMUSILL %, w u particularly important are increased branching of oligosaccharides and increased expression ofN-acetylgalactosamine-containing oligosaccharides. The presence of larger, N-linked oligosaccharides in tumour cells in general is well documented [Dennis et al., Cancer cells: 1989 (1); pp 87-93] and arises because of increases in ss (1-6) N-acetylglucosamnine type branches on N-linked oligosaccharides. This was shown by Takasaki et al., who demonstrated an increase in tri-and tetra-antennary N-

linked oligosaccharides in cancer cells [Takasaki et al., Methods in Enzymology 1978 (50) ; pp. 50-54]. ss (I-6) N-acetylglucosamine branching is particularly common, and it is now accepted that ss (I-6) branching is a feature of tumorigenesis.

Mommers et ai. [International Journal of Cancer, Oct 22nd 1999 (vol. 84, &num;5) ; pp. 466-9] reported that the mucin glycoprotein MUC1 showed aberrant expression in DCIS and Invasive Breast Cancer cells compared to normal cells. Mucins are 0linked glycoproteins. This finding implies that those lesions with aberrant expression are at a higher risk of developing subsequent invasive breast carcinoma. However, this work was carried out using immunohistochemical staining of tissue sections by monoclonal antibodies and was only applied to a specific 0-linked glycoprotein.

Schnitt et al., (Virchows Arch. 1995 (vol. 427 &num;3) ; pp. 251-258) also studied mucins in DCIS and Invasive Breast Cancer cells using immunohistochemical techniques.

Surprisingly, we have found that by using normal phase High Performance Liquid Chromatography, the total glycosylation profile of a DCIS cell is a good indicator of whether the cell has an invasive carcinoma phenotype and, therefore, is likely to develop into an Invasive Breast Carcinoma cell. We have found that certain oligosaccharides are more prevalent in cells with an invasive phenotype and that these oligosaccharides are characteristic of N-linked oligosaccharides.

Thus, in a first aspect, the present invention provides a method for establishing the presence or absence of tumour cells having an invasive phenotype in a sample, comprising analysing oligosaccharides obtained from said sample, the presence of discontinuous higher molecular weight oligosaccharides being indicative of the presence of invasive, or potentially invasive, cells in the sample.

Analysis is preferably by normal phase (hydrophilic-interaction) HPLC, although it will be appreciated that any analytical method capable of showing the weighted spread of oligosaccharides will suffice.

Essentially, in the benign DCIS cells, on an HPLC trace, there are no marked peaks for oligosaccharides above about 60 minutes whereas, in the case of invasive cells, there is a number of marked peaks, especially around 75 minutes, and a broader range between about 95 and 115 minutes. It will be appreciated that the detection of oligosaccharides, and the time at which they elute, will be determined according to the processes used to prepare the oligosaccharides, the conditions under which the normal phase HPLC is run and the sensitivity of the detection system used. The times quoted above, for example, are appropriate for the conditions applying in the accompanying Example. It will be readily apparent to one skilled in the art how to apply these observations to differing conditions. For example, the graphs obtained will have a broad similarity, with benign cells exhibiting a range of early peaks which tail off and exhibit no further peaks thereafter.

It will be appreciated that the term"discontinuous"is indicative of the line observed in the graph, or trace, with that for benign cells not exhibiting any marked peaks or troughs of any statistical significance. This is particularly illustrated in

accompanying Figure 1, where the lower line, after 60 minutes, is that for the DCIS cells, the upper line showing a number of marked peaks and, thereby, being discontinuous.

It will also be appreciated from Figure 1 that the first significant oligosaccharide which appears to be indicative of invasion, or the potential for invasion, is located at around the 60 minute mark, and is generally observed along with the peaks noted above.

Figure 2 highlights the range of peaks indicative of invasion, or the potential for invasion. Isolation of the oligosaccharides represented by these peaks will enable the preparation of suitable monoclonal antibodies to bind with these oligosaccharides and, thereby, to bind with cells having the invasive phenotype. Such targeted antibodies can be used in techniques well knov/n in the art to treat or mark the cancer.

For example, the antibodies may simply mark the cells for complement activation, or they may carry suitable toxins against the cells. Alternatively, they may be used in characterisation techniques, such as visualisation in immunohistochemical staining, for example, especially where the cancer has metastasised, or spread to other tissues.

Isolation of the oligosaccharides characterised in Figure 2 will also enable the differentiation between benign cells and invasive cells, thereby allowing the preparation of monoclonal antibodies that distinguish between the two types of cell, and which may broadly be used in a similar manner to that described above.

The present invention further provides a kit for the detection of invasive cells in a sample, comprising means to prepare free oligosaccharides from the sample and control oligosaccharides indicative of invasive phenotype cells. If desired, the kit may also comprise samples to calibrate the HPLC to be used, suitable samples including fetuin sugars and/or a dextran hydrosylate preparation.

In general, it is desired to separate and purify the oligosaccharides from the sample, in order to avoid confusing the results obtained with any contaminants.

However, where mass screening is desired to be effected, this can be impractical and, under such circumstances, it is desirable to minimise the amount of steps required.

A simple method for preparing oligosaccharides in the present invention is to disrupt the cells of the sample and collect the membrane fraction, such as by centrifugation, and, subsequently, to prepare oligosaccharides from the membrane fraction by such a process as hydrazinolysis, and then to run the free oligosaccharides, which may be prepared from the membranes by centrifugation, on HPLC.

Even more simply, the cells of the sample may be gently macerated in order to disrupt cell-cell adhesion, but to generally avoid cell disruption, washed, and then treated to dissociate oligosaccharides therefrom. The preparation can then be spun down, or allowed to settle, and the clear fraction, containing the oligosaccharides, can then be run on HPLC, as above.

Alternatively, tissues that are collected for Pathological diagnosis and fixed and processed in the normal way can be used to extract the structures of interest, as shown in the Example.

The presence of positive and negative controls in the kit can then enable the sample to be run to determine the presence or absence of invasive cells. Ideally, but not essential to the invention, the method of diagnosis will involve two samples from the patient, one of which comprises disease-free tissue, for comparison.

It will be appreciated that the presence or absence of large oligosaccharides is generally indicative of the presence or absence of cells having an invasive phenotype, but that, where the tumour is in the process of becoming invasive, the boundaries may not be clear. In such circumstances, the skilled physician may then need to supplement the diagnosis provided by the present invention and/or take appropriate action, such as mastectomy.

It will be appreciated that, while the present invention applies generally to a benign tumour, it applies particularly to DCIS, and its transformation into the invasive and, potentially, metastatic condition.

The present invention further provides a method for identifying those tumour cells, which although benign, possess an invasive phenotype and are, therefore, likely to develop into invasive cancer cells, resulting in a potentially life-threatening condition.

According to an alternative aspect of the present invention, there is provided a method of characterising tumour cells in a sample, said method comprising; i) dissociating oligosaccharides from the surface of cells in the sample; ii) separating said oligosaccharides by normal phase High Performance Liquid Chromatography; iii) determining the glycosylation profile of said oligosaccharides; and iv) comparing said glycosylation profile with known standard profiles of benign and invasive carcinoma cells, in order to establish the phenotype of said

fii tumour cells.

The present invention is generally applicable to the characterisation of carcinoma cells, especially apparent (or early) carcinoma cells. However, the present invention is of particular advantage in characterising ductal carcinoma and other in situ breast carcinoma cells. The method is generally applicable to mammalian cells and, more particularly, human cells, with human breast cells, especially human DCIS cells being preferred targets for analysis or diagnosis.

According to the present invention, cells can be identified as having an invasive phenotype by analysing the glycosylation profile of the oligosaccharides removed from the cells and comparing the profile with standards from cells known to have an invasive phenotype or known to have an a non-invasive phenotype.

Application of the method of the invention shows that the profiles of cells with invasive phenotypes include different, prevalent glycofonns, compared with benign cells not having an invasive phenotype. In particular, the glycoforms displayed by the cells having invasive phenotypes are larger than those from cells which do not have an invasive phenotype.

In its broadest aspect, the present invention provides for the analysis of total oligosaccharide associated with the cell surface of cells in a sample. Although not essential to the present invention, where possible, it is generally preferred to target those oligosaccharides associated with glycoproteins on the cell surface, and particularly N-linked oligosaccharides, as it is believed that these are the members where the change in diversity is seen, and which are diagnostic of the potential for invasion.

It is possible to rank results obtained by HPLC in terms of their glucose unit values, and this is facilitated by the calibration of the apparatus using a dextran ladder.

Suitable dextran ladders are commercially available. The glycoforms present at higher levels in the cells with invasive phenotypes can generally be grouped according to their glucose unit values. Characteristic glucose unit values are higher than those associated with benign cells, and are typically those associated with the peaks described above. It will also be appreciated that cancers vary from patient to patient, so that any one or more of the characteristic peaks or glucose unit value groups is generally sufficient to diagnose the invasive phenotype.

When using the procedure detailed in the accompanying Example, the first identifiable peak that is specific to cells with an invasive phenotype falls within a range of 1.9 to 2.56 glucose units (95% confidence interval) in DCIS cells with an invasive phenotype. Thus, the invention provides a method as defined above, wherein the presence of a statistically significant peak having a glucose unit value of 1.9 or above is diagnostic of the presence of cells having an invasive phenotype.

It will be appreciated that, when applied to a breast tissue sample, the present invention is capable of identifying cells which, when identified as having an invasive phenotype, can be classified as Invasive Breast Cancer cells.

The present invention further provides for the use in the manufacture of a medicament for the treatment of cancer which medicament is characterised in that it comprises an element capable of binding to glycoproteins which display oligosaccharides identified as being present in, or on, cells with an invasive phenotype. The element may bind to the protein part of the glycoprotein, but more

preferably to the oligosaccharide displayed by the glycoprotein. The element may then act as a marker or may affect the activity or function of the glycoprotein or of the cell.

The present invention also provides for a kit of parts for screening, diagnosis or prognosis of the stage or seventy of cancer, or for assessing the success of treatment of said disease, or for predicting the onset of invasive cancer in a patient, by assessing the glycosylation profile of cells and identifying whether the cells have an invasive phenotype. In general, such a kit may comprise parts suitable for carrying out the following: i) a first step of separating said proteins of interest by HPLC; ii) a further step comprising comparing said expression pattern with known standard profiles of benign and invasive carcinomas, wherein proteins showing larger glycofbrms are diagnostic of invasive carcinoma.

In general, cells having an invasive phenotype have been shown to have a characteristic glycosylation pattern. To establish this pattern, oligosaccharides are removed from cells in a sample and analysed by normal phase High Performance Liquid Chromatography. This separates oligosaccharides by molecular weight and charge, with small, uncharged oligosaccharides being eluted first. Larger, more highly charged oligosaccharides are eluted as time progresses. This results in a series of peaks relating to individual oligosaccharides defined according to their relative abundance and their relative weight (in glucose units). A dextran ladder and fetuin sugars are suitably run before the samples, as controls. Removal of oligosaccharides from cells and normal phase High Performance Liquid Chromatography are well documented in the art. It will be appreciated that other techniques for the separation and analysis of oligosaccharides, for example fluorophore-assisted electrophoresis and capillary electrophoresis may also be used to identify the invasive cancerassociated oligosaccharides.

In accompanying Figure 1, the elution profiles of DCIS and invasive tumour components are overlain on the same graph, and show certain oligosaccharides eluting in the later part of the graph to be common to samples containing invasive cells and which did not elute in DCIS samples.

A cell with an"invasive phenotype"may be part of a benign or non-invasive tumour, but is likely to develop into a malignant, invasive or metastatic cancer cell.

A"carcinoma"is a tumour that originates in the epithelia of the body.

The term"oligosaccharide"is well known in the art, and generally refers to a multimer of monosaccharide sugar units covalently linked together. N-linked oligosaccharides form at least part of the sugar component of a glycoprotein. In general, N-linked oligosaccharides have a common tri-mannosyl core, and contain an N-acetylglucosamine residue at their reducing termini, by which residue they are covalently linked to the amide group of an asparagine residue on the protein.

Glycoproteins are proteins that display at least one sugar residue on their surface. The sugar moiety is generally an oligosaccharide. The term"glycofbrm", as used herein, refers to different oligosaccharide conformations that can be displayed by the same protein. Different glycoforms of the same glycoprotein may have differing molecular weights and charges due to the different oligosaccharide component displayed by each glycoprotein. The protein components however, will remain the same in each individual. Larger glycoforms are those glycoproteins which have a larger oligosaccharide component than the oligosaccharide component of another glycoform.

By"total oligosaccharide", it will be understood that this refers to as much oligosaccharide as is prepared by any one analytical procedure. For example, if oligosaccharides are prepared from non-disrupted cells, then only oligosaccharides present on the cell surface will be analysed, and then only those separated by the procedure chosen. In general, it is preferred to prepare all cell surface oligosaccharides.

Oligosaccharides may be prepared from the protein component of a glycoprotein by procedures well known to those skilled in the art. For example, oligosaccharides may be cleaved from the protein by hydrolysis, hydrazinolysis, by the action of an acid, base, detergent or enzyme, or by mechanical means.

A"standard profile", as used herein, refers to the glycosylation profile obtained from cells that have been taken from tumours that are known to be benign or metastatic and, therefore, exhibit characteristic glycosylation profiles.

The following Example illustrates the present invention.

EXAMPLE In preparation for this study, seven breast tumour specimens were selected which contained both areas of DCIS and areas of invasive tumour.

Each formalin fixed paraffin-wax embedded specimen was cut to prepare slides for oligosaccharide release. 6x 1 O ! lm sections were cut for oligosaccharide release and were captured on acid washed prepared slides. During all aspects of laboratory work it was essential that starch-free gloves were worn at all times and that work was carried out on clean sheets of paper to ensure no environmental carbohydrate was picked up.

Microdissection and preparation for oligosaccharide release The lOum sections were dewaxed by baking for twenty minutes in a 60 C oven followed by 2x3 minutes in xylene, 2x1 minute in 100% alcohol and 1 minute in 70% alcohol. The sections were then microdissected using a Swann Morton sterile surgical blade 12D under a light microscope using a x4 objective.

For each patient, three slides were dissected to retain only areas of DCIS and the remaining 2 (or 3) slides dissected to retain only invasive tumour.

The slides containing the cells of interest, either DCIS or invasive tumour cells, were cut using a diamond knife to enable them to fit into reactor vials which were then placed on a freeze drier for 48 hours. 2 x 2.5mg fetuin (A and B) from

foetal calf serum were included as positive controls for the oligosaccharide release step. Fetuin sugars are known to contain both 0-linked oligosaccharides and large N- linked sialylated oligosaccharides.

Chemical release of oligosaccharides by hydrazinolysis Samples were subjected to hydrazinolysis, a process allowing N-linked and 0-linked oligosaccharides to be released intact from the protein backbone through cleavage at asparagine or serine/threonine (Kobata A. , Tools for glycobiology- principles of glycobiology. Oxford Glycosystems: 1994, pages 3-11).

The procedure adopted for all the samples was as follows: 1) 800nI anhydrous hydrazine was added to each sample followed by a blanket of argon gas to displace the oxygen; 2) Samples were carefully shaken and put in a hot block set at 950C for 16 and a half hours; 3) After removal and allowing to cool, the hydrazine reaction mixture for each sample was combined in round bottomed flasks and dried down by rotary evaporation; 4) Each sample was washed three times with 1 ml toluene, combined in the flask and dried each time; 5) 2 ml saturated sodium bicarbonate was added to each flask before incubating on ice for one hour; 6) All free primary amino groups were acetylated through addition of 30gel acetic anhydride. Samples were mixed well and this procedure repeated four times to ensure complete re-N-acetylation.

The following steps were then undertaken: 7) Cation exchange columns were prepared in Polyprep columns from Bio-rad. 2mls

Dowex AG50 (H+form) resin was put in each column. Columns were then washed with ten column volumes of HPLC water. 8) Samples were passed through the columns to desalt them, followed by washing with four column volumes of HPLC water.

9) Washings and samples were collected in test tubes. The samples were dried immediately after aliquotting into 1.5ml Eppendorf tubes and their lids pierced.

Most were dried on the centrifugal evaporator though this only held 200 tubes.

The remaining Eppendorf tubes were flash frozen in liquid nitrogen and then dried on a freeze drier.

Paper chromatography to remove peptides and collect oligosaccharides All samples were subjected to descending paper chromatography, using butanol: ethanol: water (4: 1: 1) as the mobile phase, to remove any remaining salts and peptides from the oligosaccharide pool. The Whatman filters used in this process may contain environmental carbohydrate and so a filter control was included whereby no sample was loaded but the washings were collected.

1) 1 o l HPLC water was added to each Eppendorf tube which was then vortexed and centrifuged to ensure the residue had dissolved.

2) The solution was spotted onto Whatman 3M chromatography paper, cut into strips, 5p1 was spotted at a time and allowed to dry, before the next amount was added.

3) Strips were placed in the chromatography tank, and subjected to descending chromatography for 48 hours using butanol: ethanol: water (4: 1: 1 by volume) as the mobile phase.

4) Strips were then air dried, and the origin containing the oligosaccharides cut out and transferred to a 2ml plastic Luer lock syringe attached to a 0. 2 ! lm filter. The syringe was placed in a plastic centrifuge tube and the paper wetted-out by soaking in Iml methanol, forlO minutes the methanol was removed from the paper by centrifugation in a Centaur bench-top centrifuge at 3000 rpm for 10 minutes.

5) lml HPLC water was added to elute the oligosaccharides from the paper and the process repeated. This water step was then repeated a second time.

6) Each test tube therefore contained 3ml of glycan solution which was aliquotted into 2xl. 5ml eppendorfs and dried on the centrifugal evaporator.

Labelling glycans with 2-AB 2-AB is a fluorescent'tag'which links to each oligosaccharide at its reducing terminus allowing sensitive detection and analysis. Labelling occurs through a reductive amination reaction. The labelled glycans have the same stability as those which are unlabelled [Bigge et al., Analytical biochemistry: 1995 (230) ; 229-238] and the oligosaccharide structure is unaffected.

1) 0. 3ml acetic acid and 0.7 ml dimethyl sulphoxide was added to 0.048 g 2-AB and the resulting solution (solution A) vortexed.

2) 0.5ml of solution A was added to 0.031 g sodium cyanoborohydride resulting in solution B. 5 ! l1 of solution B was added to each eppendorf.

3) The Eppendorf tubes containing the oligosaccharides and the 2-AB label were mixed, centrifuged and placed in a hot block preset to 65 C for two hours.

4) After cooling, samples were subjected to descending paper chromatography on Whatman 3M paper for 2 hours using the same method as previously described.

This step was included to remove any unconjugated 2-AB from the samples.

5) The samples and washings were collected in test tubes, aliquotted into 1. 5ml Eppendorf tubes and dried on the centrifugal evaporator.

Oligosaccharide analysis using normal phase HPLC Analysis was carried out using a normal-phase chromatography (GlycoSep N) column. This resolves both neutral and charged oligosaccharide species. The column was calibrated using a 2-AB labelled dextran hydrolysate'ladder'such that elution of the oligosaccharides in the sample could be expressed in terms of glucose unit value.

A column oven was used and set to 30 C.

Oligosaccharides were detected on elution using a Jasco FP920 fluorimeter, excitation 330nm, emission 420 nm. The buffer system used was a descending acetonitrile gradient containing 250mM ammonium formate buffer pH 4.4.

Buffer preparation was as follows: Buffer preparation - 250mM ammonium formate pH 4.4 :

1) pH machine was calibrated with fresh solution of pH 7 and pH 4.

2) 27.8 ml ammonia was added to 972. 2ml HPLC water in a large beaker on a stirrer in the fume hood.

3) Solution was adjusted to pH 4.4 with formic acid using the pH meter.

4) This had made up a 500mM solution which was then diluted using HPLC water to make up the required 250mM solution.

Buffer gradient used:

0 minutes-20% ammonium formate 0 minutes-0. 4 ml/min 132 minutes-53% ammonium formate 135 minutes-100% ammonium formate 135 minutes-0. 4 ml/min 137 minutes - 100% ammonium formate 142 minutes-100% ammonium formate 145 minutes-20% ammonium formate 180 minutes-20% ammonium formate 180 minutes - 0. 4 mllmin 181 minutes-20% ammonium formate 181 minutes-0 ml/min Overall, 45.7 ml 100% acetonitrile and 26.5 ml ammonium formate per sample.

This programme was loaded onto the Gilson HPLC system.

Prior to analysis, the labelled oligosaccharides were reconstituted with 120 ! l1 of buffer (70% acetonitrile, 30% HPLC water) vortexed, centrifuged and aliquotted into autosampler vials. The samples were injected onto the column using a Jasco autosampler.

Data collection and analysis was onto a bench-top PC through the Borwin data handling programme.

Results Normal phase (hydrophilic-interaction) HPLC separation resolves oligosaccharides according to their size and their charge. Small, uncharged species are those which elute after a short amount of time, as time progresses, larger, increasingly more charged oligosaccharides are eluted.

At the start of each HPLC programme, a dextran'ladder'was analysed. From its resulting profile, glucose unit values were assigned to each peak. A calibration curve was then plotted for the glucose unit value against elution time-a MicroCal Origin scientific data-analysis programme (version 5) was used for this purpose. This allowed any oligosaccharide peak in the samples to be read off the standard curve and assigned a glucose unit value.

A filter control profile was obtained to show how much of the sample profiles was due simply to the environmental carbohydrate contained within the Whatman filters used.

Fetuin sugars were also analysed to show the retention times of mono-, bi-, triand tetra-sialylated species so that sample oligosaccharide profiles could be interpreted.

From this, an obvious difference between the invasive and DCIS profiles was seen. There was a more diverse range of oligosaccharides present in the invasive than in the DCIS samples. The elution profiles of DCIS and invasive tumour components overlain on the same graph showed certain oligosaccharides eluting in the later part of the graph to be common to almost all of the invasive samples and yet these did not elute in most of the DCIS samples. This is shown in accompanying Figure 1.

The first oligosaccharide elevated in the invasive profiles is that indicated as "1"in Figure 1. The mean glucose unit value for this peak using all invasive samples was 2.29. Using a glucose unit value tolerance curve generated by running 30 dextran ladder samples through the column, the 95% confidence interval was 1. 9-2. 56 glucose units.

The peaks for each oligosaccharide in the sample were integrated and the area under the curve (, ut x minutes) calculated. The total area of all oligosaccharides was then calculated for each sample and, therefore, the % that each oligosaccharide contributed to the total oligosaccharide pool could be determined.

The % of the total oligosaccharide pool eluting at or after a glucose unit value of 1.9 was calculated. The difference in the amount of oligosaccharides greater than 1.9 glucose units in size in the invasive cancer cells compared with the in situ cells was found to be significant (Student paired t-test p=0. 042).

Having found that the oligosaccharides greater than or equal to 1.9 glucose unit value were present in increased levels in invasive breast cancer cells, further analysis found certain groups of oligosaccharides to be of particular interest. These oligosaccharides were present in almost all of the invasive cancer cell extracts. These oligosacharide groups are marked on Figure 2.

The dextran hydrolysate ladder and calibration curve obtained were standard, similar to those obtained in previous studies, indicating that the machinery was running correctly.

The fetuin sugars showed that oligosaccharides were released intact, the filter control profile had some small oligosaccharides eluting early in the run, the fluorescence being lower by a factor of 10 than the dextran ladder profile. Three main oligosaccharides eluted. No filter control oligosaccharides eluted after 60 minutes, therefore all sample peaks eluting after this time were entirely due to the breast cancer cell oligosaccharides. This has particular relevance to the invasive tumour profiles.

In 6 of the 7 samples few, if any, oligosaccharides eluted from the DCIS cells in the latter half of the run whereas the equivalent invasive cell profile did have groups of oligosaccharide peaks eluting after this time. This difference was remarkably obvious and consistent and after calculating the appropriate glucose unit value cut off point of 1.9, was found to be significant (Student paired t-test p=0. 042).

The results show that the breast cancer cells which have invaded through the stroma express large oligosaccharides not present on cells of DCIS. This suggests that these oligosaccharides are involved in the process of tumour progression and, when expressed by DCIS cells are diagnostic of imminent cell motility or invasion or, indeed, that invasion has already started.

Claims

Claims :

1. A method for establishing the presence or absence of tumour cells having an invasive phenotype in a sample, comprising analysing oligosaccharides obtained from said sample, the presence of discontinuous higher molecular weight oligosaccharides being indicative of the presence of invasive, or potentially invasive, cells in the sample.

2. A method according to claim 1, wherein the presence of a statistically significant peak having a glucose unit value of 1.9 or above is diagnostic of the presence of cells having an invasive phenotype.

3. A method according to claim 1 or 2, wherein the sample is derived from human breast tissue.

4. A method according to any preceding claim, wherein analysis is by hydrophilic-interaction HPLC.

5. A method according to any preceding claim, wherein the presence of statistically significant oligosaccharide peaks at or above 60 minutes is indicative of cells having an invasive phenotype.

6. A method according to any preceding claim, wherein the presence of statistically significant oligosaccharide peaks at around 75 minutes is indicative of cells having an invasive phenotype.

7. A method according to any preceding claim, wherein the presence of statistically significant oligosaccharide peaks between 95 and 115 minutes is indicative of cells having an invasive phenotype.

8. A monoclonal antibody specific for an oligosaccharide defined in any of claims 2 or 5 to 7.

9. A monoclonal antibody specific for a protein displaying an oligosaccharide defined in any of claims 2 or 5 to 7.

10. A kit for use in association with HPLC for the detection of invasive cells in a sample in accordance with any of claims 1 to 7, comprising means to prepare free oligosaccharides from the sample and control oligosaccharides indicative of invasive phenotype cells.

11. A kit according to claim 10, further comprising samples to calibrate the HPLC to be used, such as fetuin sugars and/or a dextran hydrosylate preparation.