British Medical Bulltm (1991) Vol 47, No. 2, pp 324-342 © The Bntuh Council 1991
Practical application of determinants of cell behaviour I O Ellis Department of Histopathology, City Hospital, Nottingham, UK
During the last fifteen years there has been considerable innovation and development of techniques which can be used to study cell biology and behaviour. These include immunocytochemistry, monoclonal antibody technology, molecular biology and biomedical engineering resulting in advanced flow and image cytometric systems. The volume of research results emerging from basic scientists, pathologists and clinicians relative to these techniques is ever increasing. This generation of data demonstrates the immense interest in the potential of these methods to provide useful information of biological and clinical importance. Breast cancer is one area that has received such attention because of its frequency in western countries and its exhibition of a wide spectrum of clinical behaviour. An acceptance that conservation techniques of surgery and radiotherapy are safe alternatives to mastectomy and developments of chemotherapeutic schedules have now provided the patient, surgeon and oncologist with realistic choices of treatment. The question now being asked by many groups is whether this choice can be influenced appropriately by a knowledge of molecular processes which are present, or that have occurred, and which may influence the tumour cell.
This paper addresses, firstly, some of the techniques and reagents (Table 1) which can now be used to study the cell and secondly their potential for direct clinical application. The greatest impact in this area has been the development and application of monoclonal and polyclonal antibody technology.
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Table 1 Techniques and reagents used to study breast cancer cells Techniques
Reagents
Immunocytochemistry ELIZA Flow cytometry
Monoclonal and polyclonal antibodies Monoclonal and polyclonal antibodies Monoclonal and polyclonal antibodies Stoichiometric DNA stains Monoclonal and polyclonal antibodies Stoichiometric DNA stains Routine cytology and histology stains
Image cytometry and morphometry Recombinant DNA technology Filter hybridisation (blotting) In situ hybridisation Polymerase chain reaction
Restriction endonucleases DNA and RNA probes Restriction endonucleases DNA and RNA probes Oligonucleotide primers and DNA polymerase
MONOCLONAL AND POLYCLONAL ANTIBODIES Both polyclonal and monoclonal antibodies have been produced to breast epithelial cells, to their cell products, to tumours or to common cell determinants which have associations with breast disease. The expression of antigens identified by these antibodies can be studied either by immunocytochemistry on tissue sections or cytology preparations, by enzyme linked immunozorbant assay (ELIZA) of body fluids or soluble cell fractions and by flow cytometry of single cell or cell fraction preparations. Antigens which vary in expression or appear at specific times during normal cell growth, function, differentiation, proliferation or neoplasia are of particular interest. Epithelial mucins The primary functional role of breast epithelial cells is to produce milk during lactation. The lipid rich droplets are surrounded by a membrane derived from the epithelial cell apical membrane. This membrane is rich in carbohydrates on the external and, uniquely, on the internal cytoplasmic side. A highly immunogenic mucin component of the membrane has stimulated considerable investigation. Initially polyclonal antibodies to delipidated human milk fat globule membrane (HMFGM) 1 were produced. Following production of polyclonal antiserum many groups have produced monoclonal antisera to such high molecular weight glycoprotein mucins {see review Ref. 2) normally expressed on apical membrane of human breast and other epithelial cells. The varying reactivity of
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Table 2 Antibodies to the following molecules have stimulated the most interest in breast cancer research Class of molecule
Specific antibodies
Epithelial mucins Growth factors and receptors
Polymorphic epithelial mucin Epidermal growth factor receptor Transforming growth factors a&p c-erbB-2 ras myc Thymidine (autoradiography) Bromodeoxyuridine Ki67 Proliferating cell nuclear antigen Oestrogen & progesterone receptor Cathepsin-D Plasminogen activator P-glycoprotein
Oncogene products Cell cycle markers
Hormone receptors Enzymes Multidrug resistance gene
some of these antibodies gave the initial impression that there may be a family of such mucins present in glandular epithelial cells and led to a variety of names being used to describe the mucins recognised by particular antibodies. Examples are epithelial membrane antigen (EMA)3 and the 'MAM' series.4 Interlaboratory collaborative studies5 have shown that one highly immunogenic high molecular weight glycoprotein (over 400kD) carries most of the epitopes recognized by these antibodies. Recently this mucin has been characterized further and called 'polymorphic epithelial mucin' (PEM).6 It is a transmembrane glycoprotein with a large cytoplasmic tail which interacts with the actin-containing microfilaments. The core protein is made up of tandem repeats of 20 aminoacids and reacts with many of the antibodies raised to HMFGM and breast cancer cells. Carcinomas show a difference in glycosylation of the core protein from normal cells which could result in exposure of some epitopes in malignancy. The function of this and other HMFGM mucins is not clear but they may have a role in cell protection or lubrication.2 Other antisera to lower molecular weight blood group and oncofetal antigens7 have also been produced through immunisation with HMFGM. Growth factors and their receptors Epidermal growth factor and epidermal growth factor receptor
Epidermal growth factor (EGF) is a powerful polypeptide growth factor which is essential in the development of mammary glands
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in mice.8 It has been shown to influence, and in some situations be necessary for, the growth of normal mammary epithelium breast cancer cell lines and other cell lines.9 The mitogenic effect of EGF is mediated through binding widi a specific membrane receptor-epidermal growth factor receptor (EGFR).10 It is a 170KD transmembrane glycoprotein with a heavily glycosylated external domain responsible for ligand binding, a single transmembrane spanning sequence and an internal domain with tyrosine kinase activity.11 Activation of the receptor induces cell division usually synergistically with other growth regulatory signals. The protein sequence for EGFR has been determined and its gene cloned. The gene and receptor show close similarity to two oncogenes and their oncoproteins, namely v-erbB-1 and c-erbB-2.12 EGFRs have been found in a variety of animal and human tissues, are particularly elevated in squamous tumours of the skin13 and have been identified in a proportion of breast cancers.14 EGFR may be measured in breast tumours by radioligand binding assay of membrane fractions and by immunohistological staining of tumour sections. There is a good correlation between both methods.14 The reported frequency of expression varies between 15-60% of tumours. There is a strong relationship to tumour size14 which could influence the frequency in individual studies. Clinical interest in EGFR has been further stimulated by the demonstration of an association between EGFR expression and poor prognosis.15'16 Transforming growth factors alpha and beta
The epidermal growth factor family of growth regulators also includes transforming growth factor alpha (TGF-a), a related single chain polypeptide17 which can stimulate growth by binding to and activating the EGF receptor.18'19 In normal cells, levels of TGF-a, EGF and EGFR are regulated by oestrogens. In normal breast epithelial cell lines and some breast cancer cell lines the production of TGF-a is also controlled in part by oestrogen which stimulates TGF-a synthesis and secretion.20 It is secreted by all tumour cell lines including breast and the possibility of an autocrine regulation of growth in the presence of EGFR has been suggested.21 Transforming growth factor beta (TGF-(3) is an unrelated two chain polypeptide which is a member of a complex structurally
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related family of growth and differentiation factors.22 The various forms of TGF-P bind a set of three structurally and functionally distinct cell surface receptors. TGF-P has a growth inhibitory effect on epithelial cells including mammary epithelium.23 Some squamous cell carcinoma cell lines are not inhibited by TGF-P and it has been suggested that TGF-P may be involved in regulation of normal epithelial cell growth through negative feedback, carcinomas having altered growth due to their lack of response. The evidence that both TGF-oc and TGF-P are produced in situ has led to speculation that there is an autocrine growth loop, involving TGF-a and modulated by TGF-P, influencing cell proliferation in normal and malignant epithelial tissues.22 Oncogenes and their products The development and progression of a malignant phenotype of human tumours is related to abnormalities of structure or activity of proto-oncogenes.24 Many cellular oncogenes have been found to be activated in breast cancer. Of these, c-erbB-2, c-myc, and ras have excited the most interest. A variety of other oncogenes and their products are being actively investigated at present but their relationships to clinical variables is not yet clear. c-erbB-2 The proto-oncogene c-erbB-2 (also known as neu or HER—2) encodes a 190 kD transmembrane glycoprotein similar in structure to the epidermal growth factor receptor. The c-erbB-2 is a distinct gene but is related to the c-erbB-\ gene (epidermal growth factor receptor) and v-er6B oncogene (avian erythroblastosis virus, AEV).25 Other oncogenes of AEV include the homologene c-erbA gene, a steroid receptor gene encoding a nuclear receptor for thyroid hormone.26 In humans both c-erbA and c-erbB-2 are located on chromosome 17 p 21-22.25-26 The extracellular domains of c-erbB-2 protein and EGFR are 40% identical in sequence and both possess two regions rich in cysteine residues which may be responsible for stabilization of their three dimensional structure and ability to bind ligands. The two proteins are also identical in sequence in about 80% of their amino acids forming the intracellular tyrosine kinase domain. Ligand-like activity has been reported in the conditioned medium of ras transformed cells27 and recently a 30 kD factor has been identified as a ligand for the c-erbB-2
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receptor.28 Monoclonal antibodies which bind to and downregulate mutant c-erbB-2 receptor expression inhibit tumour cell growth in vitro and in vivo29 and overexpression of the normal c-erbB-2 protein in NIH 3T3 cells leads to transformation.30-31 Recently antibodies to natural human c-erbB-2 have been shown to inhibit the growth of the breast cancer derived cell line SKBR-3 which expresses high levels of the protein.32 These observations imply an important role for c-erbB-2 in at least a subset of human breast cancers. The c-erbB-2 gene has been found to be amplified in 15-20% of invasive human breast carcinomas11 and gene amplification of over 3 fold appears characteristically to be associated with c-erbB2 gene protein localization on tumour cell membranes. This localization can be detected by immunocytochemical techniques.33 High frequency of gene amplification of around 50% have been found in ductal carcinoma in situ (DCIS) 34 and of over 90% in Pagets disease of the nipple.35 In DCIS there is an association with the large cell comedo subtype.34 There has been increasing interest in the role of c-erbB-2 oncogene in breast cancer, particularly its relationship to prognosis.36 Overexpression of c-erbB-2 oncogene is now generally accepted to correlate with poor prognosis in both primary operable and advanced breast cancer patients by some groups, 113738 although the relationship appears in many studies to be confined to lymph node positive patients. Overexpression is also associated with poor differentiation.373940 Our knowledge of the function of c-erbB-2 oncoprotein is rudimentary. The similarities to EGFR and its persistent overexpression in a significant proportion of breast carcinomas with poorer prognosis imply an important growth regulatory role. This is further supported by the observation that monoclonal antibodies raised against the extracellular domain41 have exerted an antitumour effect on mutant neu transformed NIH 3T3 cells and on a human breast tumour derived cell line. In addition it is known that EGFR expression is associated with poorer prognosis15-16 and one might postulate that both EGFR and c-erbB-2 oncoprotein are components of a mechanism responsible for breast tumour development or progression. Indeed one group42 has demonstrated that c-erbB-2 oncoprotein can act as a substrate for EGFR tyrosine kinase and it has recently been demonstrated that a combination of expression of EGFR and c-erbB-2 more efficiently transforms cells than either protein alone.43 A possible hypothesis, of
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their role in neoplasia or tumour progression, is that binding of ligand to an increasing number of receptors leads to an elevation in phosphokinase activity which would promote cell replication. ras The ras family of genes are closely related {see review Ref. 44). They encode GTP binding proteins which act as intracellular messengers involved in transmitting signals from activated growth factor receptors to the nucleus. The most widely studied ras protein is the c-rasH p21 oncoprotein which has sequence homology with the G-protein alpha subunit involved in adenylate cyclase activation. Single or small numbers of amino acid point mutations of ras genes can induce cell transformation and have been found in approximately 15% of human carcinomas45 and ras mRNA is overexpressed in a variety of tumours including breast.46 The mutations result in gene products deficient in GTPase activity and hence may influence proliferation control. c-rasH p21 protein expression has been studied in human breast tumours by immunohistological staining. There are conflicting results with some groups finding increased p21 expression in carcinomas47 and premalignant lesions48 and others finding similar expression in benign and malignant lesions.49 The specificity of some antibodies has been questioned. At present one must conclude that ras genes may play a role in development of some human breast cancers but their practical role is questionable. c myc
c-myc is one of a number of cellular and viral oncogenes {myc, myb, fos) which code for nuclear proteins that appear to have a role in embryogenesis and proliferation. Gene amplification has been found in up to 30% of breast cancers. Its 62kDA protein product is found in the nucleus during the GO to Gl phase of the cell cycle {see review Ref. 44). In breast cancer an association has been found between c-myc protein expression and poorer histological grade of the tumour suggesting a relationship with tumour differentiation, but no association has been found with prognosis.50 Cell cycle assessment Many groups have now shown that an estimate of the proliferative activity of a tumour through whatever means can give prognostic51
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and therapeutic52 information. It must be borne in mind that true assessment of the proliferative rate of a tumour can only be determined by combining measurements of the growth fraction and the cell cycle time through sequential sampling. A single measurement in time of the growth fraction of a tumour should be regarded only as an index of proliferation.53 The growth fraction can be assessed by counting the proportion of cells in mitosis (mitotic index) but this requires high quality tissue sections. Some groups have shown that simple counting of mitotic figures can provide powerful prognostic information alone,54 or when included in a grading system.55 Recently techniques of labelling cells which are active in the cell cycle have provided alternatives. Cells in the DNA synthesis phase of the cycle (S phase) will take up thymidine or anologues of thymidine such as 5-bromodeoxyuridine (BRDU). Encorporated molecules can be identified by prior radiolabelling, or in the case of BRDU by immunocytochemistry or flow cytometry using anti-BRDU antibody. Counts of the proportion of labelled cells will give an estimate of the S phase fraction (SPF). Two further alternative growth fraction markers have emerged. The monoclonal antibody Ki67 was raised against a Hodgkins disease cell line and identifies cells active in the cell cycle. The antigen is of unknown structure and is highly labile. It can be used to estimate a 'proliferative index' in breast cancers which can provide prognostic information.56 Antibodies are now available to a 36 kD nuclear protein named Proliferating Cell Nuclear Antigen (PCNA) or cyclin which appears in the cell nucleus in late S phase. This molecule is an auxiliary protein of the DNA polymerase-d enzyme and can be identified in standard histological sections using immunohistochemistry.57 It can potentially provide information analagous but not directly comparable with flow cytometric estimation of SPF.
Hormone receptors Monoclonal antibodies raised to the nuclear receptors for oestrogen and progesterone allow both assay of cytosol fraction of tumours using ELIZA methods and in siiu localisation of receptor by immunocytochemistry. These advances have improved the accuracy of prediction of response to hormone therapy, {see Forrest, this issue.
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P-glycoprotein A proportion of tumours of many types will develop resistance to a variety of chemotherapeutic agents. This multidrug resistant phenotype is associated with expression of a 170kD membrane glycoprotein (P-glycoprotein) which acts as an energy dependent pump removing certain families of chemotherapeutic drugs.58 Antibodies to P-glycoprotein have been produced85 which can be used to identify expression in tumour samples. Few studies have been performed in human breast cancer but development of the MDR phenotype appears to be a late phenomenon59 and may therefore be of limited clinical value.
Enzymes A role in tumour cell migration and metastasis has been suggested for protease-type enzymes such as plasminogen activator60 and cathepsin-D61 which have been identified in breast tumour samples. It is implied that tumours with higher levels of these enzymes or proenzymes could have a greater ability to digest basement membrane, extracellular matrix and connective tissue components.61 Interestingly although a prognostic relationship exists between the levels of these two enzymes found in tumour tissue homogenates, this relationship appears to be independent of tumour stage60'62 which could also be considered to be a marker of metastatic potential. High levels of these enzymes are present in some inflammatory cells and these results could reflect the degree of host response to rumour, which is related to tumour differentiation. Use of more sensitive systems targeted specifically at the tumour cell should provide a more accurate assessment of the role of these enzymes in tumour cell behaviour.
Other molecules of interest The reactivity of numerous additional antibodies have been investigated in breast cancer. These include intermediate filament proteins, basement membrane components, CEA, a-lactalbumin, caseins, blood group antigens and many others (see review Ref. 63). Many of these reagents have not found wide acceptance as routine tests of clinical importance.
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MORPHOMETRY AND CYTOMETRY Objective measurements of the shape, arrangement and tinctorial characteristics of breast tumour cell populations can be made using a variety of simple morphometric principles or more complex computer assisted morphometric,64 image cytometric65 and flow cytometric equipment.66 The type of morphometric information that can be obtained includes cell and nuclear size and shape, cellularity, mitotic frequency (see 'Cell cycle assessment' above), nuclear chromatin and nucleolar texture.54'64 Use of fluorescent or visible stoichiometric DNA stains allow measurement of nuclear DNA content by flow cytometry and densitometry with image cytometry. DNA content can be used to assess abnormalities of ploidy and to estimate the SPF. Many of the variables measured by these techniques have been shown to provide prognostic information, albeit of varying importance, in breast cancer patients. 546768 MOLECULAR BIOLOGY At present the recombinant DNA techniques of filter hybridisation (blotting) and in situ hybridization techniques are providing important understanding of tumour cell biology, in particular gene regulation and gene abnormalities during neoplasia. These techniques (see review Ref. 69) utilise the ability of complimentary strands of DNA or RNA to reassociate (hybridise) to form a double stranded structure. Labelled synthetic nucleotide sequences can act as probes which will bind to their analogous DNA or RNA sequence in tissue (in situ hybridisation) or to denatured DNA or RNA preparations separated and immobilised on synthetic filters. These methods can be time consuming and probes may not be widely available and expensive to purchase. Expertise in these methods is generally confined to major research institutions and for these reasons the results are not being applied directly to clinical situations. A greater understanding of the relevance of molecular changes will be required before there is wider use of these techniques in clinical laboratories. The rapidity and diversity of results currently emerging leads one to believe that there will be a future for molecular technology in clinical management of breast disease.
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PRACTICAL APPLICATIONS Recognition of malignancy Following the discovery of monoclonal antibody technology many groups have attempted to identify tumour specific antigens. This search has been largely fruitless. It is perhaps not surprising that such molecules are either elusive or do not exist. The neoplastic cell is a product of a normal cell through a process of transformation and is likely to retain most of its basic cellular and molecular structure. Novel antigens generated by this process would most probably lead to cell elimination by the host immune response. Some groups have claimed that certain oncogene products may be increased in malignancy and in preneoplastic processes and could be used to recognize these processes.48 Some of these claims have not been substantiated by others trying to reproduce the results.49 The techniques used such as immunocytochemistry are interpreted by subjective methods and can be difficult to quantify. This may explain some of the inconsistency; at present it certainly precludes the use of such reagents in routine diagnostic situations. Finally this approach, unless combined with odier molecular investigations, identifies specific individual events which only occur in a proportion of tumours and could not therefore be applied to a routine system involving all tumours. However, gross disturbances in the tumour cell DNA or genome are very unlikely to occur in normal or non neoplastic cells. Identification of a highly aneuploid stem line by image or flow cytometry, or detection of high levels of amplification of oncogenes such as c-erbB-2 by molecular techniques or immunocytochemistry could be used to identify malignancy for a subset of tumours. These tumours are usually easily recognized by pathologists in histological sections or cytological preparations as they tend to exhibit characteristic morphological features of malignancy. There is far greater need for accurate identification of preneoplastic conditions and malignant tumours at an early stage of development where detection methods such as mammography, and cytological and histological diagnosis rely on very subtle changes. At present the only technique which has shown any real potential as a routine system to aid diagnosis is image cytometry. By combined assessment of nuclear morphology, DNA content and possibly chromatin texture, relatively acceptable levels of sensitivity and specificity for diagnosis of malignancy can be obtained.70 Further development of preparative techniques, system hardware
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and feature selection criteria are likely and some believe that this technology could assist traditional cytology in preoperative diagnosis of breast disease. Assessment of prognosis Many of the biological processes or effects which can be examined by the above techniques are related to the prognosis of a given tumour. The mechanisms for some associations have an apparently simple basis. For example, a measure of proliferative activity can give an indication of tumour doubling time. Other associations are less clearly understood but may relate to differentiation or events occurring during tumour promotion or progression to a more anaplastic state. Clinicians and scientists are now widely beginning to appreciate the ability of traditional prognostic factors in breast cancer, such as tumour size, lymph node stage, histological grade and histological type, and are able to predict the likely prognosis of a given individual at presentation.35'71'72 When combined in a prognostic index such prognostic factors can give a highly accurate assessment of likely prognosis.73 It would be fortunate indeed if a single molecular event could offer analogous information. This would provide a relatively simple objective method of assessing prognosis. One must bear in mind that traditional factors are dependent on a host of variables including the time a tumour has been present (size and lymph node stage), differentiation (grade and type), proliferation (grade) and metastatic potential (lymph node stage). In a study using a series of multivariate analyses designed to establish its independent prognostic value in comparison with traditional factors (size, stage and grade), c-erbB-2 protein expression achieved significance only when included with the time related variables of tumour size and lymph node stage. When the powerful tumour related prognostic factor, histological grade, was introduced into the analysis the independent significance of c-erbB-2 protein expression was lost.37 Similar results have been found when EGFR expression has been analysed in a similar fashion15 and using prognostic antibodies to epithelial mucin.74 Such studies do show that if accurate information about tumour grade is not available such molecular information can provide analogous, although less powerful, information which could act as a substitute for histological grade.
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It is possible to discuss some reasons for this lack of power by again using c-erbB-2 amplification as an example, although it is difficult to speculate without precise knowledge of its function. c-erbB-2 gene amplification is found in only a small proportion of tumours and for this reason alone it is perhaps not surprising that it fails to provide prognostic information of a magnitude similar to histological grade. It has been suggested that amplification and over expression of certain genes may be reflected in tumour cell morphology75 which has been borne out partly by evidence that c-erbB-2 amplification is related to large cell morphology, particularly in ductal carcinoma in situ.34 Histological grading is assessed by combining the appearance of various morphological features and mitotic figure frequency.55 Thus it provides a summation of a variety of tumour variables. One could extrapolate further from the above tentative evidence and suggest that histological grade gives an overview of various molecular events affecting morphological appearance. It is unlikely therefore that a single molecular event could compete with histological grade in such a statistical multivariate analysis. The future of clinical application of molecular markers of prognosis will be in combination, providing information analogous to histological grade. Histological grade has been criticised for its subjective nature and lack of reproducibility, although when carried out by enthusiastic pathologists good correlation can be achieved. Use of guidelines and introduction of semiquantitative assessment of the components could improve consistency further.76 The most important component of histological grade is the assessment of mitotic frequency. Objective measurements of tumour cell proliferation such as percentage of cells in mitosis, thymidine or BRDU labelling index, S phase fraction measurement and Ki67 labelling index have all been shown to provide powerful prognostic information. Again the information is analogous to histological grade but the methods also provide a tumour-dependent prognostic variable which can be measured in an objective fashion. All have some drawbacks; mitotic frequency is time consuming to perform, thymidine and BRDU labelling indices require in vivo or in vitro incorporation and subsequent detection systems. S phase fraction requires flow cytometric equipment and Ki67 labelling index immunocytochemical staining of fresh tissue. It is difficult to justify some of these methods in a cost conscious Health Service environment when histological grade and tumour type can
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be assessed rapidly on a routine histological tissue section and provide extremely powerful information about prognosis. Response to treatment There is a need to develop accurate methods of predicting the response to primary local treatment to identify at least three groups; those at high risk of distant relapse, those who will be cured by local modalities and those who will respond to systemic therapy. Use of a prognostic index using traditional prognostic factors73 or one incorporating morphometric or molecular variables 3754 can help identify a group of patients who have an extremely good prognosis, which is not significantly different from the expected survival of die non breast cancer bearing female population. These patients clearly have a very low risk of significant local or distant recurrence and use of systemic forms of treatment, which may carry a hazard in themselves, may not be justified as a routine policy.62 Similarly a group of patients with a grave prognosis and a high risk of distant recurrence can also be identified. Use of adjuvant systemic therapy could easily be justified in these patients. In addition to the use of predictive prognostic indices there is evidence that response to chemotherapy can be predicted in patients with breast cancer through measurement of the proliferative activity of the tumour. It is widely accepted that tumours with a very high proliferative rate such as acute leukaemias, high grade lymphomas and germ cell rumours can respond dramatically to chemotherapy schedules. Similar although less dramatic behaviour has been reported in breast cancer. A relationship has been shown between S phase fraction (SPF) and tumour response in patients with stage 11-1 lla disease.52 Tumours with a low SPF ( 10%) all responded. There was considerable overlap between the groups but these results are encouraging and supportive data has emerged from thymidine labelling (TLI) studies on patients receiving adjuvant chemotherapy. Long term follow up has shown that patients with a high tumour TLI had delayed recurrence in contrast to patients witfi a low tumour TLI in whom no benefit was observed.77 It is likely that use of such information about growth fraction if combined with other data such as hormone receptor status and histological information (for example histologi-
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cal type) could become an important part of the assessment of breast cancer patients. Detection of metastasis Although many markers of tumour differentiation exist currently there is no widely accepted marker present on the primary tumour cell which can indicate metastatic potential. Binding of tumour cells by Helix Pomatia lectin has been found to have an association with lymph node stage78'79 but the mechanism of this relationship is unclear and the relationship needs further evaluation. Information about the potential or the reality of lymph node or distant metastasis can only be obtained reliably through tissue biopsy. Clinical examination, detection of highly elevated levels of certain markers in serum and imaging techniques can be used to detect metastatic disease in more advanced stages. Deposits over 2 cm in size can sometimes be visualized by imaging using radiolabeled targeting monoclonal antibodies6380 but this method is not sensitive enough to detect small early deposits. Antibodies to epithelial antigens such as cytokeratins and epithelial mucins can also be used to detect micrometastases in excised lymph nodes or bone marrow aspirate samples by immunocytochemical staining. This increases the detection rate of metastic disease from levels found by routine histologicaJ examination. The long term significance of such findings is still debated but there is evidence from the few published studies that patients with micrometastatic disease have a higher chance of subsequent overt recurrence.81
CONCLUSION The relatively recent developments described above are dramatic. They provide mechanisms and evidence to increase our understanding of the biology and behaviour of breast cancer and have served to stimulate and bring together scientists, pathologists, oncologists and surgeons working on this common condition. At present the direa applications to laboratory and clinical practice are limited but there is no doubt that such information will be applied more rigorously to the clinical setting resulting in a greater awareness of their potential in the management of the individual patient.
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21 Sporn M, Roberts AB. Autocrine growth factors and cancer. Nature 1985; 313: 747-751 22 Partridge M, Green M, Langdon J, Feldmann M. Production TGF-a and TGF-P by cultured keratinocytes, skin and oral squamous cell carcinomas— potential autocrine regulation of normal and malignant epithelial cell proliferation. Br J Cancer 1989; 60: 542-548 23 Silberstein GB, Daniel CW. Reversible inhibition of mammary gland growth by transforming growth factor p. Science 1987; 237: 291 24 Slamon DJ, deKerion JB, Verma IM, Cline MJ. Expression of cellular oncogenes in human malignancies. Science 1984; 224: 256-262 25 Yamamoto T, Ikawa S, Akiyama T, et al. Similarity of protein encoded by the human c-erbB-2 gene to epidermal growth factor receptor. Nature 1986; 319: 230-234 26 Weinberger C, Thompson CC, Ong ES, Lebo R, Gruol DJ, Evans RM. The c-erbA gene encodes a thyroid hormone receptor. Nature 1986; 324: 641 27 Yarden Y, Weinberg RA. Experimental approaches to hypothetical hormones: detection of a candidate ligand of the neu proto-oncogcne. Proc Nad Acad Sci USA 1989; 86: 3179-3183 28 Lupu R, Colomer R, Zugmaier G, et al. Direct interaction of a ligand for the erbB2 oncogene product with the EGF receptor and pl85" bB2 . Science 1990; 249: 1552-1555 29 Maguire HC, Greene MI. The neu (c-erbB-2) oncogene. Seminars in Oncology 1989; 16: 148-155 30 Hudziak RM, Schlessinger J, Ullrich A. Increased expression of the putative growth factor receptor pl85HER2 causes transformation and tumourigenesis of NIH 3T3 cells. Proc Nad Acad Sci USA 1987; 84: 7159-7163 31 Fiore PPD, Pierce JH, Kraus MH, Segatto O, King CR, Aaranson SA. erbB2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science 1987; 237: 178-182 32 Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, Ullrich A. pl85HER2 monoclonal antibody has antiproliferadve effects in vitro and sensitizes human breast rumour cells to tumour necrosis factor. Mol Cell Biol 1989; 9: 1165-1172 33 Venter DJ, Kumar S, Tuzi N, Gulhck WJ. Overexpression of the c-erbB-2 oncoprotein in human breast carcinomas: Immunohistochemical assessment correlated with gene amplificadon. Lancet 1987; (ii): 69-71 34 Van de Vijver MJ, Peterse JL, Mooi WJ, et al. Neu-protein overexpression in breast cancer: association with comedo-type ductal carcinoma in situ and limited prognostic value in stage II breast cancer. N Engl J Med 1988; 319: 12391245 35 Lammie GA, Barnes DM, Millis RR, Gullick WJ. An immunohistochemical study of the presence of c-erbB-2 protein in Pagct's disease of the nipple. Histopathology 1989; 15: 505-514 36 Barnes DM. Editorial: Breast cancer and a proto-oncogenc. Br Med J 1989; 299: 1061 37 Lovekin C, Ellis IO, Locker A, et al. c-erbB-2 oncoprotein expression in primary and advanced breast cancer. Br J Cancer 1990 (In press) 38 Slamon D, Godolphin W, Jones L, et al. Studies of the HER-2/ncu protooncogene in human breast and ovarian cancer. Science 1989; 244: 707-712 39 Wright C, Angus B, Nicholson S, et al. Expression of c-erbB-2 oncoprotein: A prognostic marker in human breast cancer. Cancer Res 1989; 49: 2087-2091 40 Barnes DM, Lammie GA, Millis RR, Gullick WJ, Allen DS, Altman DG. An immunohistochemical evaluation of c-erbB-2 expression in human breast carcinoma. Br J Cancer 1988; 58: 448-^152 41 Drebin JA, Link VC, Weinberg RA, Greene MI. Inhibition of tumour growth
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by a monoclonal antibody reactive with an oncogene encoded tumour antigen. Proc Natl Acad Sri USA 1986; 83: 9129-9133 Kadowaki T, Kasuga M, Tobe K, et al. A Mw 190,000 glycoprotein phosphorylated on tyrosine residues in Epidermal Growth Factor Receptor stimulated KB cells is the product of c-erbB-2 gene. Biochem Biophys Res Commun 1987; 144: 699 Kokai Y, Myers J, Wada T, et al. Synergistic interaction of pl85c neu and the EFG receptor leads to transformation of rodent fibroblasts. Cell 1989; 58: 287-292 Gelmann E, Lippman M. Understanding the role of oncogenes in human breast cancer. In: Sluyser M ed. Growth factors and oncogenes in breast cancer. VCH Publishers, 1987. Weinberg RA. The action of oncogenes in the cytoplasm and nucleus. Science 1985; 230: 770-774 Slamdn DJ, deKerion JB, Verma IM, Cline MJ. Expression of cellular oncogenes in human malignancies. Science 1984; 224: 256-262 Walker RA, Wilkinson N. p21 ras protein expression in benign and malignant human breast. J Pathol 1988; 156: 147-153 Ohuchi N, Thor A, Page DL, Hand PH, Haeter S, Schlom J. Expression of the 21000 molecular weight ras protein in a spectrum of benign and malignant human mammary tissues. Cancer Res 1986; 46: 2511-2519 Ghosh AK, Moore M, Harris M. Immunohistochemical detection of ras oncogene p21 product in benign and malignant mammary tissue in man. J Clin Pathol 1986; 39: 428-434 Locker A, Dowle C, Ellis I, et al. C-myc oncogene product expression and prognosis in operable breast cancer. Br J Cancer 1989; 60: 669-672 Meyer J, Friedman L. Prediction of early course of breast carcinoma by thymidine labelling. Cancer 1983; 51: 1879-1886 Remvikos Y, Beuzebocp P, Zajdela A, ct al. Correlation of proliferative activity of breast cancer with the response to cytotoxic chemotherapy. J Nat Cancer Inst 1989; 81(18): 1383-1387 Wright NA. Cell proliferation in health and disease. In: Anthony PP, MacSween RNM ed. Recent advances in Histopathology. Edinburgh: Churchill Livingstone, 1984: pp. 17-34 Baak JPA. Morphometry and breast cancer. Human Pathology 1990 (In press) Elston C. Grading of invasive carcinoma of the breast. In: Page DL, Anderson TJ ed. Diagnostic Histopathology of the Breast. Edinburgh; Churchill Livingstone, 1987; 300-311 Gerdcs J, Lcllc RJ, Pickartz H, et al. Growth fractions in breast cancers determined in situ with monoclonal antibody Ki-67. J Clin Pathol 1986; 39: 977-980 Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-PCNA/Cyclin monoclonal antibodies in fixed embedded tissues. Am J Pathol 1989; 134: 733-739 Morrow CS, Cowan KH. Mechanisms and clinical significance of multidrug resistance. Oncology 1988; 2(10): 55-64 Schneider J, Bak M, Efferth T, Kaufmann M, Mattern J, Volm M. P-Glycoprotein expression in treated and untreated human breast cancer. Br J Cancer 1989; 60: 815-818 Duffy MJ, O'Grady P, Devaney D, O'Siorain L, Fennelly JJ, Lijnen HR. Tissue-type plasminogen activator, a new prognostic marker in breast cancer. Cancer Res 1988; 48: 1348-1349 Rochefort H, Augereau P, Capony F, et al. The 52K cathepsin-D of breast cancer: Structure, regulation, functionand clinical value. In: Lippman ME, Dickson RB ed. Breast Cancer: Cellular and Molecular Biology. Boston: Kluwer Acad., 1988: pp. 207-222 McGuire WL, Tandon AK, Allred DC, Chamness GC, Clark GM. How to
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use prognostic factors in axillary node-negative breast cancer patients. J Nat Cancer Inst 1990; 82(12): 1006-1015 Sterns EE, Cochran AJ. Monoclonal antibodies in the diagnosis and treatment of carcinoma of the breast. Surg Gynaecol Obstet 1989; 169: 81-98 Baak JPA, Oort J. A manual of morphometry in diagnostic pathology. Berlin: Springer, 1983. Brugal G. Analysis of microscopic preparations. In: Jasmin G, Proschek L, ed. Methods and achievements of experimental pathology. Basel: Karger, 1984: 1-33 HedJey D, Friedlander M, Taylor I, Gugg C, Musgrove L. Method for analysis of cellular DNA content of paraffin embedded pathological material using flow cytomctry. J Histochem Cytochem 1983; 31: 1333 Auer G, Eriksson E, Azavedo E, Casperson T, Wallgren A. Prognostic significance of nuclear DNA content in mammary adenocarcinoma in humans. Cancer Res 1984; 44: 394-396 Walker R, Camplejohn R. DNA Flow cytometry of human breast carcinomas and its relationship to transferrin and epidermal growth factor receptors. J Pathol 1986; 150: 37-42 Goudic RB. DNA technology in histopathology. In: Anthony P, MacSween R ed. Recent Advances in Histopathology. Edinburgh: Churchill Livingstone, 1989: 1-21 Locker AP, Dilks B, Gilmour A, Ellis IO, Elston CW, Blarney RW. Aspiration cytology diagnosis of breast lesions by nuclear DNA content. Br J Surg 1990; 77(6): A707 Blarney RW, Davics CJ, Elston CW, Johnson J, Haybittle JL, Maynard, PV. Prognostic factors in breast cancer—the formation of a prognostic index. Clin Oncol 1979; 5: 227-236 Page DL, Anderson TJ, Sakamoto G. Infiltrating carcinoma: major histological types. In Page DL, Anderson TJ, eds. Diagnostic Histopathology of the Breast. Edinburgh: Churchill Livingstone, 1987: pp. 193-235 Todd J, Dowle C, Williams M et al. Confirmation of a prognostic index in primary breast cancer. Br J Cancer 1987; 56: 489-492 Ellis I, Bell J, Todd J, et al. Evaluation of immunoreactivity with monoclonal antibody NCRC11 in breast carcinoma. Br J Cancer 1987; 56: 295-299 Cardiff RD. Cellular and molecular aspects of neoplastic progression in the mammary gland. Eur J Cancer Clin Oncol 1988; 24: 15-20 Royal, College, of, Pathologists, Working, Group. Pathology Reporting in Breast Cancer Screening. NHS Breast Screening Programme Publication, 1990. Tubiana M, Pejovic MH, Koscielny S, et al. Growth rate, kinetics of tumour cell proliferation and long term outcome in human breast cancer. Int J Cancer 1989; 44: 17-22 Leathern AJ, Brooks SA. Predictive value of lectin binding on breast cancer recurrence and survival. Lancet 1987; i: 1054-1056 Fenlon S, Ellis I, Bell J, Todd J, Elston C, Blarney R. Helix Pomatia and Ulex Europeus lectin binding in human breast cancer. J Pathol 1987; 152: 169-176 Rainsbury RM. The localisation of mammary tumours by labelled monoclonal antibodies. Br J Surg 1984; 71: 805-812 International, (Ludwig), Breast, Cancer, Study, Group. Prognostic importance of occult axillary lymph node micrometastases from breast cancers. Lancet 1990; 335: 1565-1568
Practical application of determinants of cell behaviour I O Ellis Department of Histopathology, City Hospital, Nottingham, UK
During the last fifteen years there has been considerable innovation and development of techniques which can be used to study cell biology and behaviour. These include immunocytochemistry, monoclonal antibody technology, molecular biology and biomedical engineering resulting in advanced flow and image cytometric systems. The volume of research results emerging from basic scientists, pathologists and clinicians relative to these techniques is ever increasing. This generation of data demonstrates the immense interest in the potential of these methods to provide useful information of biological and clinical importance. Breast cancer is one area that has received such attention because of its frequency in western countries and its exhibition of a wide spectrum of clinical behaviour. An acceptance that conservation techniques of surgery and radiotherapy are safe alternatives to mastectomy and developments of chemotherapeutic schedules have now provided the patient, surgeon and oncologist with realistic choices of treatment. The question now being asked by many groups is whether this choice can be influenced appropriately by a knowledge of molecular processes which are present, or that have occurred, and which may influence the tumour cell.
This paper addresses, firstly, some of the techniques and reagents (Table 1) which can now be used to study the cell and secondly their potential for direct clinical application. The greatest impact in this area has been the development and application of monoclonal and polyclonal antibody technology.
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Table 1 Techniques and reagents used to study breast cancer cells Techniques
Reagents
Immunocytochemistry ELIZA Flow cytometry
Monoclonal and polyclonal antibodies Monoclonal and polyclonal antibodies Monoclonal and polyclonal antibodies Stoichiometric DNA stains Monoclonal and polyclonal antibodies Stoichiometric DNA stains Routine cytology and histology stains
Image cytometry and morphometry Recombinant DNA technology Filter hybridisation (blotting) In situ hybridisation Polymerase chain reaction
Restriction endonucleases DNA and RNA probes Restriction endonucleases DNA and RNA probes Oligonucleotide primers and DNA polymerase
MONOCLONAL AND POLYCLONAL ANTIBODIES Both polyclonal and monoclonal antibodies have been produced to breast epithelial cells, to their cell products, to tumours or to common cell determinants which have associations with breast disease. The expression of antigens identified by these antibodies can be studied either by immunocytochemistry on tissue sections or cytology preparations, by enzyme linked immunozorbant assay (ELIZA) of body fluids or soluble cell fractions and by flow cytometry of single cell or cell fraction preparations. Antigens which vary in expression or appear at specific times during normal cell growth, function, differentiation, proliferation or neoplasia are of particular interest. Epithelial mucins The primary functional role of breast epithelial cells is to produce milk during lactation. The lipid rich droplets are surrounded by a membrane derived from the epithelial cell apical membrane. This membrane is rich in carbohydrates on the external and, uniquely, on the internal cytoplasmic side. A highly immunogenic mucin component of the membrane has stimulated considerable investigation. Initially polyclonal antibodies to delipidated human milk fat globule membrane (HMFGM) 1 were produced. Following production of polyclonal antiserum many groups have produced monoclonal antisera to such high molecular weight glycoprotein mucins {see review Ref. 2) normally expressed on apical membrane of human breast and other epithelial cells. The varying reactivity of
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Table 2 Antibodies to the following molecules have stimulated the most interest in breast cancer research Class of molecule
Specific antibodies
Epithelial mucins Growth factors and receptors
Polymorphic epithelial mucin Epidermal growth factor receptor Transforming growth factors a&p c-erbB-2 ras myc Thymidine (autoradiography) Bromodeoxyuridine Ki67 Proliferating cell nuclear antigen Oestrogen & progesterone receptor Cathepsin-D Plasminogen activator P-glycoprotein
Oncogene products Cell cycle markers
Hormone receptors Enzymes Multidrug resistance gene
some of these antibodies gave the initial impression that there may be a family of such mucins present in glandular epithelial cells and led to a variety of names being used to describe the mucins recognised by particular antibodies. Examples are epithelial membrane antigen (EMA)3 and the 'MAM' series.4 Interlaboratory collaborative studies5 have shown that one highly immunogenic high molecular weight glycoprotein (over 400kD) carries most of the epitopes recognized by these antibodies. Recently this mucin has been characterized further and called 'polymorphic epithelial mucin' (PEM).6 It is a transmembrane glycoprotein with a large cytoplasmic tail which interacts with the actin-containing microfilaments. The core protein is made up of tandem repeats of 20 aminoacids and reacts with many of the antibodies raised to HMFGM and breast cancer cells. Carcinomas show a difference in glycosylation of the core protein from normal cells which could result in exposure of some epitopes in malignancy. The function of this and other HMFGM mucins is not clear but they may have a role in cell protection or lubrication.2 Other antisera to lower molecular weight blood group and oncofetal antigens7 have also been produced through immunisation with HMFGM. Growth factors and their receptors Epidermal growth factor and epidermal growth factor receptor
Epidermal growth factor (EGF) is a powerful polypeptide growth factor which is essential in the development of mammary glands
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in mice.8 It has been shown to influence, and in some situations be necessary for, the growth of normal mammary epithelium breast cancer cell lines and other cell lines.9 The mitogenic effect of EGF is mediated through binding widi a specific membrane receptor-epidermal growth factor receptor (EGFR).10 It is a 170KD transmembrane glycoprotein with a heavily glycosylated external domain responsible for ligand binding, a single transmembrane spanning sequence and an internal domain with tyrosine kinase activity.11 Activation of the receptor induces cell division usually synergistically with other growth regulatory signals. The protein sequence for EGFR has been determined and its gene cloned. The gene and receptor show close similarity to two oncogenes and their oncoproteins, namely v-erbB-1 and c-erbB-2.12 EGFRs have been found in a variety of animal and human tissues, are particularly elevated in squamous tumours of the skin13 and have been identified in a proportion of breast cancers.14 EGFR may be measured in breast tumours by radioligand binding assay of membrane fractions and by immunohistological staining of tumour sections. There is a good correlation between both methods.14 The reported frequency of expression varies between 15-60% of tumours. There is a strong relationship to tumour size14 which could influence the frequency in individual studies. Clinical interest in EGFR has been further stimulated by the demonstration of an association between EGFR expression and poor prognosis.15'16 Transforming growth factors alpha and beta
The epidermal growth factor family of growth regulators also includes transforming growth factor alpha (TGF-a), a related single chain polypeptide17 which can stimulate growth by binding to and activating the EGF receptor.18'19 In normal cells, levels of TGF-a, EGF and EGFR are regulated by oestrogens. In normal breast epithelial cell lines and some breast cancer cell lines the production of TGF-a is also controlled in part by oestrogen which stimulates TGF-a synthesis and secretion.20 It is secreted by all tumour cell lines including breast and the possibility of an autocrine regulation of growth in the presence of EGFR has been suggested.21 Transforming growth factor beta (TGF-(3) is an unrelated two chain polypeptide which is a member of a complex structurally
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related family of growth and differentiation factors.22 The various forms of TGF-P bind a set of three structurally and functionally distinct cell surface receptors. TGF-P has a growth inhibitory effect on epithelial cells including mammary epithelium.23 Some squamous cell carcinoma cell lines are not inhibited by TGF-P and it has been suggested that TGF-P may be involved in regulation of normal epithelial cell growth through negative feedback, carcinomas having altered growth due to their lack of response. The evidence that both TGF-oc and TGF-P are produced in situ has led to speculation that there is an autocrine growth loop, involving TGF-a and modulated by TGF-P, influencing cell proliferation in normal and malignant epithelial tissues.22 Oncogenes and their products The development and progression of a malignant phenotype of human tumours is related to abnormalities of structure or activity of proto-oncogenes.24 Many cellular oncogenes have been found to be activated in breast cancer. Of these, c-erbB-2, c-myc, and ras have excited the most interest. A variety of other oncogenes and their products are being actively investigated at present but their relationships to clinical variables is not yet clear. c-erbB-2 The proto-oncogene c-erbB-2 (also known as neu or HER—2) encodes a 190 kD transmembrane glycoprotein similar in structure to the epidermal growth factor receptor. The c-erbB-2 is a distinct gene but is related to the c-erbB-\ gene (epidermal growth factor receptor) and v-er6B oncogene (avian erythroblastosis virus, AEV).25 Other oncogenes of AEV include the homologene c-erbA gene, a steroid receptor gene encoding a nuclear receptor for thyroid hormone.26 In humans both c-erbA and c-erbB-2 are located on chromosome 17 p 21-22.25-26 The extracellular domains of c-erbB-2 protein and EGFR are 40% identical in sequence and both possess two regions rich in cysteine residues which may be responsible for stabilization of their three dimensional structure and ability to bind ligands. The two proteins are also identical in sequence in about 80% of their amino acids forming the intracellular tyrosine kinase domain. Ligand-like activity has been reported in the conditioned medium of ras transformed cells27 and recently a 30 kD factor has been identified as a ligand for the c-erbB-2
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receptor.28 Monoclonal antibodies which bind to and downregulate mutant c-erbB-2 receptor expression inhibit tumour cell growth in vitro and in vivo29 and overexpression of the normal c-erbB-2 protein in NIH 3T3 cells leads to transformation.30-31 Recently antibodies to natural human c-erbB-2 have been shown to inhibit the growth of the breast cancer derived cell line SKBR-3 which expresses high levels of the protein.32 These observations imply an important role for c-erbB-2 in at least a subset of human breast cancers. The c-erbB-2 gene has been found to be amplified in 15-20% of invasive human breast carcinomas11 and gene amplification of over 3 fold appears characteristically to be associated with c-erbB2 gene protein localization on tumour cell membranes. This localization can be detected by immunocytochemical techniques.33 High frequency of gene amplification of around 50% have been found in ductal carcinoma in situ (DCIS) 34 and of over 90% in Pagets disease of the nipple.35 In DCIS there is an association with the large cell comedo subtype.34 There has been increasing interest in the role of c-erbB-2 oncogene in breast cancer, particularly its relationship to prognosis.36 Overexpression of c-erbB-2 oncogene is now generally accepted to correlate with poor prognosis in both primary operable and advanced breast cancer patients by some groups, 113738 although the relationship appears in many studies to be confined to lymph node positive patients. Overexpression is also associated with poor differentiation.373940 Our knowledge of the function of c-erbB-2 oncoprotein is rudimentary. The similarities to EGFR and its persistent overexpression in a significant proportion of breast carcinomas with poorer prognosis imply an important growth regulatory role. This is further supported by the observation that monoclonal antibodies raised against the extracellular domain41 have exerted an antitumour effect on mutant neu transformed NIH 3T3 cells and on a human breast tumour derived cell line. In addition it is known that EGFR expression is associated with poorer prognosis15-16 and one might postulate that both EGFR and c-erbB-2 oncoprotein are components of a mechanism responsible for breast tumour development or progression. Indeed one group42 has demonstrated that c-erbB-2 oncoprotein can act as a substrate for EGFR tyrosine kinase and it has recently been demonstrated that a combination of expression of EGFR and c-erbB-2 more efficiently transforms cells than either protein alone.43 A possible hypothesis, of
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their role in neoplasia or tumour progression, is that binding of ligand to an increasing number of receptors leads to an elevation in phosphokinase activity which would promote cell replication. ras The ras family of genes are closely related {see review Ref. 44). They encode GTP binding proteins which act as intracellular messengers involved in transmitting signals from activated growth factor receptors to the nucleus. The most widely studied ras protein is the c-rasH p21 oncoprotein which has sequence homology with the G-protein alpha subunit involved in adenylate cyclase activation. Single or small numbers of amino acid point mutations of ras genes can induce cell transformation and have been found in approximately 15% of human carcinomas45 and ras mRNA is overexpressed in a variety of tumours including breast.46 The mutations result in gene products deficient in GTPase activity and hence may influence proliferation control. c-rasH p21 protein expression has been studied in human breast tumours by immunohistological staining. There are conflicting results with some groups finding increased p21 expression in carcinomas47 and premalignant lesions48 and others finding similar expression in benign and malignant lesions.49 The specificity of some antibodies has been questioned. At present one must conclude that ras genes may play a role in development of some human breast cancers but their practical role is questionable. c myc
c-myc is one of a number of cellular and viral oncogenes {myc, myb, fos) which code for nuclear proteins that appear to have a role in embryogenesis and proliferation. Gene amplification has been found in up to 30% of breast cancers. Its 62kDA protein product is found in the nucleus during the GO to Gl phase of the cell cycle {see review Ref. 44). In breast cancer an association has been found between c-myc protein expression and poorer histological grade of the tumour suggesting a relationship with tumour differentiation, but no association has been found with prognosis.50 Cell cycle assessment Many groups have now shown that an estimate of the proliferative activity of a tumour through whatever means can give prognostic51
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and therapeutic52 information. It must be borne in mind that true assessment of the proliferative rate of a tumour can only be determined by combining measurements of the growth fraction and the cell cycle time through sequential sampling. A single measurement in time of the growth fraction of a tumour should be regarded only as an index of proliferation.53 The growth fraction can be assessed by counting the proportion of cells in mitosis (mitotic index) but this requires high quality tissue sections. Some groups have shown that simple counting of mitotic figures can provide powerful prognostic information alone,54 or when included in a grading system.55 Recently techniques of labelling cells which are active in the cell cycle have provided alternatives. Cells in the DNA synthesis phase of the cycle (S phase) will take up thymidine or anologues of thymidine such as 5-bromodeoxyuridine (BRDU). Encorporated molecules can be identified by prior radiolabelling, or in the case of BRDU by immunocytochemistry or flow cytometry using anti-BRDU antibody. Counts of the proportion of labelled cells will give an estimate of the S phase fraction (SPF). Two further alternative growth fraction markers have emerged. The monoclonal antibody Ki67 was raised against a Hodgkins disease cell line and identifies cells active in the cell cycle. The antigen is of unknown structure and is highly labile. It can be used to estimate a 'proliferative index' in breast cancers which can provide prognostic information.56 Antibodies are now available to a 36 kD nuclear protein named Proliferating Cell Nuclear Antigen (PCNA) or cyclin which appears in the cell nucleus in late S phase. This molecule is an auxiliary protein of the DNA polymerase-d enzyme and can be identified in standard histological sections using immunohistochemistry.57 It can potentially provide information analagous but not directly comparable with flow cytometric estimation of SPF.
Hormone receptors Monoclonal antibodies raised to the nuclear receptors for oestrogen and progesterone allow both assay of cytosol fraction of tumours using ELIZA methods and in siiu localisation of receptor by immunocytochemistry. These advances have improved the accuracy of prediction of response to hormone therapy, {see Forrest, this issue.
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P-glycoprotein A proportion of tumours of many types will develop resistance to a variety of chemotherapeutic agents. This multidrug resistant phenotype is associated with expression of a 170kD membrane glycoprotein (P-glycoprotein) which acts as an energy dependent pump removing certain families of chemotherapeutic drugs.58 Antibodies to P-glycoprotein have been produced85 which can be used to identify expression in tumour samples. Few studies have been performed in human breast cancer but development of the MDR phenotype appears to be a late phenomenon59 and may therefore be of limited clinical value.
Enzymes A role in tumour cell migration and metastasis has been suggested for protease-type enzymes such as plasminogen activator60 and cathepsin-D61 which have been identified in breast tumour samples. It is implied that tumours with higher levels of these enzymes or proenzymes could have a greater ability to digest basement membrane, extracellular matrix and connective tissue components.61 Interestingly although a prognostic relationship exists between the levels of these two enzymes found in tumour tissue homogenates, this relationship appears to be independent of tumour stage60'62 which could also be considered to be a marker of metastatic potential. High levels of these enzymes are present in some inflammatory cells and these results could reflect the degree of host response to rumour, which is related to tumour differentiation. Use of more sensitive systems targeted specifically at the tumour cell should provide a more accurate assessment of the role of these enzymes in tumour cell behaviour.
Other molecules of interest The reactivity of numerous additional antibodies have been investigated in breast cancer. These include intermediate filament proteins, basement membrane components, CEA, a-lactalbumin, caseins, blood group antigens and many others (see review Ref. 63). Many of these reagents have not found wide acceptance as routine tests of clinical importance.
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MORPHOMETRY AND CYTOMETRY Objective measurements of the shape, arrangement and tinctorial characteristics of breast tumour cell populations can be made using a variety of simple morphometric principles or more complex computer assisted morphometric,64 image cytometric65 and flow cytometric equipment.66 The type of morphometric information that can be obtained includes cell and nuclear size and shape, cellularity, mitotic frequency (see 'Cell cycle assessment' above), nuclear chromatin and nucleolar texture.54'64 Use of fluorescent or visible stoichiometric DNA stains allow measurement of nuclear DNA content by flow cytometry and densitometry with image cytometry. DNA content can be used to assess abnormalities of ploidy and to estimate the SPF. Many of the variables measured by these techniques have been shown to provide prognostic information, albeit of varying importance, in breast cancer patients. 546768 MOLECULAR BIOLOGY At present the recombinant DNA techniques of filter hybridisation (blotting) and in situ hybridization techniques are providing important understanding of tumour cell biology, in particular gene regulation and gene abnormalities during neoplasia. These techniques (see review Ref. 69) utilise the ability of complimentary strands of DNA or RNA to reassociate (hybridise) to form a double stranded structure. Labelled synthetic nucleotide sequences can act as probes which will bind to their analogous DNA or RNA sequence in tissue (in situ hybridisation) or to denatured DNA or RNA preparations separated and immobilised on synthetic filters. These methods can be time consuming and probes may not be widely available and expensive to purchase. Expertise in these methods is generally confined to major research institutions and for these reasons the results are not being applied directly to clinical situations. A greater understanding of the relevance of molecular changes will be required before there is wider use of these techniques in clinical laboratories. The rapidity and diversity of results currently emerging leads one to believe that there will be a future for molecular technology in clinical management of breast disease.
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PRACTICAL APPLICATIONS Recognition of malignancy Following the discovery of monoclonal antibody technology many groups have attempted to identify tumour specific antigens. This search has been largely fruitless. It is perhaps not surprising that such molecules are either elusive or do not exist. The neoplastic cell is a product of a normal cell through a process of transformation and is likely to retain most of its basic cellular and molecular structure. Novel antigens generated by this process would most probably lead to cell elimination by the host immune response. Some groups have claimed that certain oncogene products may be increased in malignancy and in preneoplastic processes and could be used to recognize these processes.48 Some of these claims have not been substantiated by others trying to reproduce the results.49 The techniques used such as immunocytochemistry are interpreted by subjective methods and can be difficult to quantify. This may explain some of the inconsistency; at present it certainly precludes the use of such reagents in routine diagnostic situations. Finally this approach, unless combined with odier molecular investigations, identifies specific individual events which only occur in a proportion of tumours and could not therefore be applied to a routine system involving all tumours. However, gross disturbances in the tumour cell DNA or genome are very unlikely to occur in normal or non neoplastic cells. Identification of a highly aneuploid stem line by image or flow cytometry, or detection of high levels of amplification of oncogenes such as c-erbB-2 by molecular techniques or immunocytochemistry could be used to identify malignancy for a subset of tumours. These tumours are usually easily recognized by pathologists in histological sections or cytological preparations as they tend to exhibit characteristic morphological features of malignancy. There is far greater need for accurate identification of preneoplastic conditions and malignant tumours at an early stage of development where detection methods such as mammography, and cytological and histological diagnosis rely on very subtle changes. At present the only technique which has shown any real potential as a routine system to aid diagnosis is image cytometry. By combined assessment of nuclear morphology, DNA content and possibly chromatin texture, relatively acceptable levels of sensitivity and specificity for diagnosis of malignancy can be obtained.70 Further development of preparative techniques, system hardware
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and feature selection criteria are likely and some believe that this technology could assist traditional cytology in preoperative diagnosis of breast disease. Assessment of prognosis Many of the biological processes or effects which can be examined by the above techniques are related to the prognosis of a given tumour. The mechanisms for some associations have an apparently simple basis. For example, a measure of proliferative activity can give an indication of tumour doubling time. Other associations are less clearly understood but may relate to differentiation or events occurring during tumour promotion or progression to a more anaplastic state. Clinicians and scientists are now widely beginning to appreciate the ability of traditional prognostic factors in breast cancer, such as tumour size, lymph node stage, histological grade and histological type, and are able to predict the likely prognosis of a given individual at presentation.35'71'72 When combined in a prognostic index such prognostic factors can give a highly accurate assessment of likely prognosis.73 It would be fortunate indeed if a single molecular event could offer analogous information. This would provide a relatively simple objective method of assessing prognosis. One must bear in mind that traditional factors are dependent on a host of variables including the time a tumour has been present (size and lymph node stage), differentiation (grade and type), proliferation (grade) and metastatic potential (lymph node stage). In a study using a series of multivariate analyses designed to establish its independent prognostic value in comparison with traditional factors (size, stage and grade), c-erbB-2 protein expression achieved significance only when included with the time related variables of tumour size and lymph node stage. When the powerful tumour related prognostic factor, histological grade, was introduced into the analysis the independent significance of c-erbB-2 protein expression was lost.37 Similar results have been found when EGFR expression has been analysed in a similar fashion15 and using prognostic antibodies to epithelial mucin.74 Such studies do show that if accurate information about tumour grade is not available such molecular information can provide analogous, although less powerful, information which could act as a substitute for histological grade.
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It is possible to discuss some reasons for this lack of power by again using c-erbB-2 amplification as an example, although it is difficult to speculate without precise knowledge of its function. c-erbB-2 gene amplification is found in only a small proportion of tumours and for this reason alone it is perhaps not surprising that it fails to provide prognostic information of a magnitude similar to histological grade. It has been suggested that amplification and over expression of certain genes may be reflected in tumour cell morphology75 which has been borne out partly by evidence that c-erbB-2 amplification is related to large cell morphology, particularly in ductal carcinoma in situ.34 Histological grading is assessed by combining the appearance of various morphological features and mitotic figure frequency.55 Thus it provides a summation of a variety of tumour variables. One could extrapolate further from the above tentative evidence and suggest that histological grade gives an overview of various molecular events affecting morphological appearance. It is unlikely therefore that a single molecular event could compete with histological grade in such a statistical multivariate analysis. The future of clinical application of molecular markers of prognosis will be in combination, providing information analogous to histological grade. Histological grade has been criticised for its subjective nature and lack of reproducibility, although when carried out by enthusiastic pathologists good correlation can be achieved. Use of guidelines and introduction of semiquantitative assessment of the components could improve consistency further.76 The most important component of histological grade is the assessment of mitotic frequency. Objective measurements of tumour cell proliferation such as percentage of cells in mitosis, thymidine or BRDU labelling index, S phase fraction measurement and Ki67 labelling index have all been shown to provide powerful prognostic information. Again the information is analogous to histological grade but the methods also provide a tumour-dependent prognostic variable which can be measured in an objective fashion. All have some drawbacks; mitotic frequency is time consuming to perform, thymidine and BRDU labelling indices require in vivo or in vitro incorporation and subsequent detection systems. S phase fraction requires flow cytometric equipment and Ki67 labelling index immunocytochemical staining of fresh tissue. It is difficult to justify some of these methods in a cost conscious Health Service environment when histological grade and tumour type can
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be assessed rapidly on a routine histological tissue section and provide extremely powerful information about prognosis. Response to treatment There is a need to develop accurate methods of predicting the response to primary local treatment to identify at least three groups; those at high risk of distant relapse, those who will be cured by local modalities and those who will respond to systemic therapy. Use of a prognostic index using traditional prognostic factors73 or one incorporating morphometric or molecular variables 3754 can help identify a group of patients who have an extremely good prognosis, which is not significantly different from the expected survival of die non breast cancer bearing female population. These patients clearly have a very low risk of significant local or distant recurrence and use of systemic forms of treatment, which may carry a hazard in themselves, may not be justified as a routine policy.62 Similarly a group of patients with a grave prognosis and a high risk of distant recurrence can also be identified. Use of adjuvant systemic therapy could easily be justified in these patients. In addition to the use of predictive prognostic indices there is evidence that response to chemotherapy can be predicted in patients with breast cancer through measurement of the proliferative activity of the tumour. It is widely accepted that tumours with a very high proliferative rate such as acute leukaemias, high grade lymphomas and germ cell rumours can respond dramatically to chemotherapy schedules. Similar although less dramatic behaviour has been reported in breast cancer. A relationship has been shown between S phase fraction (SPF) and tumour response in patients with stage 11-1 lla disease.52 Tumours with a low SPF ( 10%) all responded. There was considerable overlap between the groups but these results are encouraging and supportive data has emerged from thymidine labelling (TLI) studies on patients receiving adjuvant chemotherapy. Long term follow up has shown that patients with a high tumour TLI had delayed recurrence in contrast to patients witfi a low tumour TLI in whom no benefit was observed.77 It is likely that use of such information about growth fraction if combined with other data such as hormone receptor status and histological information (for example histologi-
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cal type) could become an important part of the assessment of breast cancer patients. Detection of metastasis Although many markers of tumour differentiation exist currently there is no widely accepted marker present on the primary tumour cell which can indicate metastatic potential. Binding of tumour cells by Helix Pomatia lectin has been found to have an association with lymph node stage78'79 but the mechanism of this relationship is unclear and the relationship needs further evaluation. Information about the potential or the reality of lymph node or distant metastasis can only be obtained reliably through tissue biopsy. Clinical examination, detection of highly elevated levels of certain markers in serum and imaging techniques can be used to detect metastatic disease in more advanced stages. Deposits over 2 cm in size can sometimes be visualized by imaging using radiolabeled targeting monoclonal antibodies6380 but this method is not sensitive enough to detect small early deposits. Antibodies to epithelial antigens such as cytokeratins and epithelial mucins can also be used to detect micrometastases in excised lymph nodes or bone marrow aspirate samples by immunocytochemical staining. This increases the detection rate of metastic disease from levels found by routine histologicaJ examination. The long term significance of such findings is still debated but there is evidence from the few published studies that patients with micrometastatic disease have a higher chance of subsequent overt recurrence.81
CONCLUSION The relatively recent developments described above are dramatic. They provide mechanisms and evidence to increase our understanding of the biology and behaviour of breast cancer and have served to stimulate and bring together scientists, pathologists, oncologists and surgeons working on this common condition. At present the direa applications to laboratory and clinical practice are limited but there is no doubt that such information will be applied more rigorously to the clinical setting resulting in a greater awareness of their potential in the management of the individual patient.
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