Guidelines on the standards for the training of specialised health professionals dealing with breast cancer.

According to EUSOMA position paper 'The requirements of a specialist breast unit', each breast unit should have a core team made up of health professionals who have undergone specialist training in breast cancer. In this paper, on behalf of EUSOMA, authors have identified the standards of training in breast cancer, to harmonise and foster breast care training in Europe. The aim of this paper is to contribute to the increase in the level of care in a breast unit, as the input of qualified health professionals increases the quality of breast cancer patient care.

Matrix metalloproteinase expression patterns in luminal A type breast carcinomas.

OBJECTIVE: Aberrant expression of individual matrix metalloproteinases has been associated with poor prognosis in various human carcinomas. The current study aimed at defining an RNA expression profile of various MMPs in breast cancer and correlating their expression with clinicopathological parameters. METHODS: The RNA expression patterns of 6 MMPs (MMP2, MMP8, MMP9, MMP10, MMP11, MMP13) were determined in 25 breast carcinomas using quantitative RT-PCR and correlated with clinicopathological parameters, including menopausal status, tumor size and grade, and lymph node involvement. RESULTS: We observed high MMP2 levels more frequently in premenopausal than in postmenopausal women (p=0.02). Analysis of luminal A type invasive ductal carcinomas (19/25), revealed an even stronger association of MMP2 with menopausal status (p=0.005). Within this subgroup, we also found a correlation between MMP11 and menopausal status (p=0.02). No correlation was found between MMP expressions and other clinicopathological parameters. In co-expression analyses MMP2-MMP10 and MMP8-MMP9 showed a weak correlation of their expression. CONCLUSIONS: Although this is a pilot study, our findings indicate that luminal A invasive ductal carcinomas commonly express high MMP2 and MMP11 levels in premenopausal breast cancer patients and suggest a co-regulation of MMP2-MMP10 and MMP8-MMP9.

Plasma gelatinase levels in patients with primary breast cancer in relation to axillary lymph node status, Her2/neu expression and other clinicopathological variables.

Matrix metalloproteinases (MMPs), in particular the gelatinases MMP2 and MMP9, are important mediators of tumour invasion and metastasis. We examined whether plasma gelatinase levels could predict lymph node metastasis in breast cancer patients. Further, we investigated the relationship of plasma gelatinase levels with Her2/neu expression, recently acknowledged as an important prognostic factor for recurrence, and with various clinicopathological factors. Preoperative plasma samples from 81 breast cancer patients were collected. Total and active gelatinase levels were measured by enzyme immunoassays and activity assays, respectively. Neither total nor active plasma MMP2 levels correlated with nodal status or with any of the classical clinicopathological factors including histological tumour type, tumour size and grade and hormone receptor status. Patients with Her2/neu overexpressing tumours showed an increase of 27% (P=0.007) in plasma MMP2 activity, but not in total MMP2, compared with patients without overexpression. MMP9 levels, total and active, did not correlate with any of the investigated variables. In contrast to MMP9, total MMP2 levels correlated significantly with active MMP2 levels. In summary, total and active plasma gelatinase levels failed to identify high risk for axillary lymph node metastasis. Active plasma MMP2 was significantly increased in patients with Her2/neu overexpressing tumours, suggesting a role for Her2/neu in the signalling pathways of MMP2 activation in carcinogenesis. However, this increase was too small to be of clinical use. Furthermore, no relationship was found between plasma gelatinase levels, total or active, and any of the clinicopathological prognostic factors.

Association between tumour characteristics and HER-2/neu by immunohistochemistry in 1362 women with primary operable breast cancer.

AIMS: To investigate the association between tumour characteristics and HER-2/neu by immunohistochemistry in primary operable breast cancer. METHODS: The association between HER-2/neu and other clinicopathological factors was evaluated in 1362 consecutive patients with primary breast cancer treated between 2000 and July 2003 in one centre. Microscopic tumour size, tumour grade, lymph node status, patient's age, oestrogen receptor (ER), progesterone receptor (PR), and joint ER/PR status were evaluated, using the chi2 test for univariate analysis and logistic regression for multivariate analysis. The hormone receptors and HER-2/neu were studied immunohistochemically. Using the HER-2/neu DAKO scoring system, scores of 0, 1+, or 2+ were defined as negative and 3+ as positive. Data for DAKO scores 2+/3+ versus 0/1+ are also presented. RESULTS: Hormone receptor negative breast cancers were more often HER-2/neu positive than hormone receptor positive cancers, both for ER (28.7% v 6.8%) and PR (19.9% v 5.9%). In multivariate analysis, both ER, PR, and tumour grade were independently associated with HER-2/neu. In ER+ tumours, HER-2/neu overexpression was significantly lower in PR+ than in PR- cases (11.5% v 5.4%). HER-2/neu overexpression (2.7%) was lowest in the large subgroup of ER+PR+ tumours with low tumour grade (grade 1-2), comprising 46.1% of all patients. CONCLUSIONS: ER, PR, and tumour grade are independent predictors for HER-2/neu overexpression in women with primary operable breast cancer. ER and PR are negatively associated with HER-2/neu, whereas tumour grade is positively associated with HER-2/neu. In women with ER+ tumours, PR status also affects the likelihood of HER-2/neu expression.

Vascular targeting of solid tumours: a major 'inverse' volume-response relationship following combretastatin A-4 phosphate treatment of rat rhabdomyosarcomas.

Tumour-specific vascularisation may be therapeutically approached in two different ways: by antiangiogenic treatments specifically directed to dividing and migrating endothelial cells, or by agents that target principally the inadequate and ill-structured tumour vasculature. Combretastatin A-4 phosphate (combreAp), a recently synthesised prodrug (OXiGENE, Lund, Sweden), is a vascular targeting agent of the latter kind. We evaluated the effect of a single intraperitoneal (i.p.) combreAp injection on the growth of rhabdomyosarcomas syngeneic in WAG/Rij rats. Different tumour volume groups, ranging between 0.1 and 27 cm(3), were selected to assess the relationship between the size at treatment time and the response to combreAp. A double combreAp treatment (2x25 mg/kg) was investigated within the same overall aim: the relationship between growth delay and tumour size. Our results show that the systemic administration of combreAp induces a clear-cut differential growth delay in the solid rat rhabdomyosarcomas: with very large tumours (>/= 14 cm(3)), a 17.6-fold stronger effect was measured than with very small tumours (<1 cm(3)). This is the 'inverse' of the volume-response seen with the conventional therapeutic approaches (radiotherapy, chemotherapy or surgery). These combreAp antitumour responses were observed without treatment limiting systemic toxicity in the rats. With clinical digital subtraction angiography, using microsurgical cannulation of a major tumour draining vessel, and with histopathology, we demonstrate that growth delay is related to an early (within 3-6 h) and extensive breakdown of tumour blood vessels. The experiments involving a second injection also indicate a volume-dependent effect of combreAp in reducing the regrowth rate of small or large rhabdomyosarcomas. This significant differential volume-response obtained with 'selective' vascular targeting, stronger in larger tumours than smaller ones, suggests the potential of broadening the therapeutic window.

Causes of inconsistency in diagnosing and classifying intraductal proliferations of the breast. European Commission Working Group on Breast Screening Pathology.

It is now widely recognised that classifying ductal carcinoma in situ (DCIS) of the breast and diagnosing atypical ductal hyperplasia are associated with significant interobserver variation. Two possible reasons for this inconsistency are differences in the interpretation of specified histological features and field selection where morphology is heterogeneous. In order to investigate the relative contribution of these two factors to inconsistent interpretation of intraductal proliferations, histological sections of 32 lesions were sent to 23 European pathologists followed 3 years later by images of small parts of these sections. Kappa statistics for diagnosing hyperplasia of usual type, atypical ductal hyperplasia and ductal carcinoma in situ were 0.54, 0.35 and 0.78 for sections and 0.47, 0.29 and 0.78 for images, respectively, showing that most of the inconsistency is due to differences in morphological interpretation. Improvements can thus be expected only if diagnostic criteria or methodology are changed. In contrast, kappa for classifying DCIS by growth pattern was very low at 0.23 for sections and better at 0.47 for images, reflecting the widely recognised variation in the growth pattern of DCIS. Higher kappa statistics were obtained when any mention of an individual growth pattern was included in that category, thus allowing multiple categories per case; but kappa was still higher for images than sections. Classifying DCIS by nuclear grade gave kappa values of 0.36 for sections and 0.49 for images, indicating that intralesional heterogeneity has hitherto been underestimated as a cause of inconsistency in classifying DCIS by this method. More rigorous assessment of the proportions of the different nuclear grades present could lead to an improvement in consistency.

Pathological work-up of sentinel lymph nodes in breast cancer. Review of current data to be considered for the formulation of guidelines.

Controversies and inconsistencies regarding the pathological work-up of sentinel lymph nodes (SNs) led the European Working Group for Breast Screening Pathology (EWGBSP) to review published data and current evidence that can promote the formulation of European guidelines for the pathological work-up of SNs. After an evaluation of the accuracy of SN biopsy as a staging procedure, the yields of different sectioning methods and the immunohistochemical detection of metastatic cells are reviewed. Currently published data do not allow the significance of micrometastases or isolated tumour cells to be established, but it is suggested that approximately 18% of the cases may be associated with further nodal (non-SN) metastases, i.e. approximately 2% of all patients initially staged by SN biopsy. The methods for the intraoperative and molecular assessment of SNs are also surveyed.

The source-skin distance measuring bridge: a method to avoid radiation teleangiectasia in the skin after interstitial therapy for breast cancer.

Inappropriate positioning of interstitial Iridium 192 implants, used as booster dose in the breast conserving treatment of mammary cancer, may cause disturbing teleangiectasia of the breast skin, when high radiation doses are delivered on the dermal blood vessels. Based on the localization of the vascular plexuses in human breast skin, and on the dose distribution around different types of interstitial implants, a method is described to avoid overlap between the high dose area of the implant and the blood vessels in the skin. The latter are demonstrated to run within the first 5 mm under the epiderm. For source lengths varying from 5 to 8 cm, simple mathematical relations exist between the maximal security margin (MSM) and intersource distance (E) for single plane implants (MSM = 0.4 (E + 1)), double plane square implants (MSM = 0.4 E) and double plane triangular implants (MSM = 0.4 (E - 1)). We developed a device to measure precisely the distance between the radioactive wires and the overlying skin, along the whole source trajectory. Using this method, the occurrence of teleangiectasia in the breast skin after interstitial implants with Ir 192 may be significantly reduced.

Merkel cell tumor of the skin: an immunohistochemical study.

Skin biopsy specimens from 12 elderly patients with Merkel cell tumors were investigated. Conventional light microscopy and immunohistochemical techniques were used. All of the tumors had similar morphologic features. Immunoreactivity for neuronspecific enolase, gastrin, calcitonin, and epithelial membrane-like antigen was demonstrated, and both neurofilaments and keratin filaments were observed. The immunohistochemical findings supported a Merkel cell origin for these Merkel cell tumors. The co-expression of neuroendocrine and epithelial markers in Merkel cell carcinomas is suggestive of neuroendocrine differentiation in a neoplasm of epithelial origin. Merkel cell carcinomas share many characteristics with neuroendocrine tumors of the bronchopulmonary and gastrointestinal tracts. All of these neoplasms may originate from cells of similar types that are present in several organs.

Consistency achieved by 23 European pathologists in categorizing ductal carcinoma in situ of the breast using five classifications. European Commission Working Group on Breast Screening Pathology.

The increased detection of ductal carcinoma in situ (DCIS) by mammographic screening, the greater use of breast-conserving surgery, and the recognition that certain histological subtypes are associated with a greater risk of local recurrence has led to the formulation of several new classifications of DCIS in recent years. There are, however, no data concerning the degree of consistency with which these schemes can be applied by reasonable numbers of pathologists. Thirty-three cases of DCIS were thus examined by a working group of 23 European pathologists who categorized them using five recently published classifications: (1) that of the European Pathologists' Working Group based on differentiation (a combination of nuclear grade and cell polarization) with categories of poorly, intermediately, and well differentiated; (2) one based entirely on nuclear grade with categories of high, intermediate, and low, currently in use in the UK national and EC-funded breast screening programs; (3) the same classification in which only two categories, high nuclear grade and other, were used; (4) the Van Nuys system in which lesions are divided into high grade, non-high grade with necrosis and non-high grade without necrosis; and (5) a two-category classification based entirely on the presence or absence of comedo necrosis. Of the three systems with three categories, Van Nuys gave the highest overall kappa statistic of 0.42. Others gave similar values of 0.37 and 0.35 showing that assessing cell polarization in addition to nuclear grade neither improves nor worsens consistency. In all three systems, the middle category was associated with the lowest value for kappa. Of the two systems with two categories, that based on nuclear grade gave the highest overall kappa of 0.46 and that based on comedo necrosis the lowest of 0.34. The most robust histological features were thus high- and low-grade nuclei and necrosis as long as the latter did not involve the recognition of a comedo growth pattern. These values probably represent the maximum achievable, at least by reasonable numbers of pathologists in everyday practice. They are better than those previously reported for classification based entirely on architecture, but further improvement is needed.

A study of the value of p53, HER2, and Bcl-2 in the prediction of response to doxorubicin and paclitaxel as single agents in metastatic breast cancer: a companion study to EORTC 10923.

This study assesses the potential value of the tumor markers p53, HER2, and Bcl-2 in predicting the clinical response to doxorubicin and paclitaxel as single agents in the treatment of metastatic breast cancer. The primary tumors of 114 patients in the European Organization for Research and Treatment of Cancer 10923 trial were assessed by immunohistochemistry using monoclonal antibodies; the results were correlated with clinical response to therapy. HER2 was positive in 24% of patients, p53 was positive in 25% of patients, and Bcl-2 was positive in 49% of patients. There was no correlation between the expression of any of the markers and the clinical response to either agent. Although methodologically limited, this study does not support the use of p53, HER2, or Bcl-2 to assist the selection of anthracycline versus taxane in metastatic breast cancer.

Breast cancer screening pathology: an assessment of the practise and needs in Belgium and Luxembourg.

Education and quality assurance (QA) in breast screening pathology have been encouraged by the Europe Against Cancer programme. As a prerequisite for the set-up of a QA programme in Belgium and in the Grand Duchy of Luxembourg, an inquiry was initiated to evaluate the daily practise in breast pathology, the modalities in handling and analysing breast specimens and the willingness of the pathologists to participate in a QA scheme. Of the 278 mailed questionnaires, 109 confidential and valid questionnaires were returned, meaning a participation rate of 40%. All 109 respondents indicated their willingness to voluntarily participate in the further QA programme. Segmental resections for conservative surgery and excision biopsies ranked first and second, respectively, in examination requests. Of the respondents, 50% complained about the lack of clinical information on the pathology request form. A multidisciplinary team approach for the diagnosis of screen-detected lesions was deemed desirable by 87% of the respondents, but only 16% of them actually participate in such pre-operative meetings. Even more puzzling is that 75% of the respondents report regular unavailability of the control radiogram of the surgical specimen removed for non-palpable lesions. One-quarter to one-third of the pathologists still regularly perform frozen sections on microcalcifications or tumours smaller than 1 cm. However, 81% of the respondents estimate that pre-operative diagnosis is not appropriate for this type of lesion. The results of this inquiry show that the guidelines for the diagnosis of screen-detected breast lesions are not yet fully applied in daily practise. The development of local comprehensive breast teams involving a pathologist should improve the co-ordination between the medical disciplines, represent an important way of disseminating the guidelines on breast screening pathology and stimulate the relay unit to conduct QA programmes.

Discrepancies in current practice of pathological evaluation of sentinel lymph nodes in breast cancer. Results of a questionnaire based survey by the European Working Group for Breast Screening Pathology.

AIMS: To evaluate aspects of the current practice of sentinel lymph node (SLN) pathology in breast cancer via a questionnaire based survey, to recognise major issues that the European guidelines for mammography screening should address in the next revision. METHODS: A questionnaire was circulated by mail or electronically by the authors in their respective countries. Replies from pathology units dealing with SLN specimens were evaluated further. RESULTS: Of the 382 respondents, 240 European pathology units were dealing with SLN specimens. Sixty per cent of these units carried out intraoperative assessment, most commonly consisting of frozen sections. Most units slice larger SLNs into pieces and only 12% assess these slices on a single haematoxylin and eosin (HE) stained slide. Seventy one per cent of the units routinely use immunohistochemistry in all cases negative by HE. The terms micrometastasis, submicrometastasis, and isolated tumour cells (ITCs) are used in 93%, 22%, and 71% of units, respectively, but have a rather heterogeneous interpretation. Molecular SLN staging was reported by only 10 units (4%). Most institutions have their own guidelines for SLN processing, but some countries also have well recognised national guidelines. CONCLUSIONS: Pathological examination of SLNs throughout Europe varies considerably and is not standardised. The European guidelines should focus on standardising examination. They should recommend techniques that identify metastases > 2 mm as a minimum standard. Uniform reporting of additional findings may also be important, because micrometastases and ITCs may in the future be shown to have clinical relevance.

Consistency of staining and reporting of oestrogen receptor immunocytochemistry within the European Union--an inter-laboratory study.

To assess the variability of oestrogen receptor (ER) testing using immunocytochemistry, centrally stained and unstained slides from breast cancers were circulated to the members of the European Working Group for Breast Screening Pathology, who were asked to report on both slides. The results showed that there was almost complete concordance among readers (kappa=0.95) in ER-negative tumours on the stained slide and excellent concordance among readers (kappa=0.82) on the slides stained in each individual laboratory. Tumours showing strong positivity were reasonably well assessed (kappa=0.57 and 0.4, respectively), but there was less concordance in tumours with moderate and low levels of ER, especially when these were heterogeneous in their staining. Because of the variation, the Working Group recommends that laboratories performing these stains should take part in a external quality assurance scheme for immunocytochemistry, should include a tumour with low ER levels as a weak positive control and should audit the percentage positive tumours in their laboratory against the accepted norms annually. The Quick score method of receptor assessment may also have too many categories for good concordance, and grouping of these into fewer categories may remove some of the variation among laboratories.

Diagnostic fine-needle aspiration cytology and immunocytochemistry analysis of a primary thyroid lymphoma presenting as an anatomic emergency.

The case of a 66-year-old woman with rapidly progressive respiratory distress caused by a massive anterior neck mass with tracheal compression is presented. Within 24 hours, fine-needle aspiration cytology (FNAC) and immunocytochemistry provided a diagnosis of high-grade B-cell lymphoma and the opportunity to institute a chemotherapeutic regimen resulting in a rapid volume reduction and airway expansion. One year after combined modality treatment the patient was in complete remission (with an estimated thyroid volume of 4 cm3). This case report illustrates the advantages of FNAC and immunocytochemistry in the diagnosis of thyroid lymphoma.

Epidermal Langerhans cells, dermal dendritic cells, and keratinocytes in viral lesions of skin and mucous membranes: an immunohistochemical study.

We wanted to evaluate the eventual expression of viral antigens and MHC class II products by keratinocytes as well as the alterations of epidermal Langerhans cells and dermal dendritic cells in viral lesions of skin and mucous membranes. Therefore we investigated 68 biopsy specimens of protracted viral lesions, such as warts, condylomas, and mollusca contagiosa, and of rapidly resolving viral lesions such as herpes simplex virus infection. For this we used immunohistochemical staining techniques and several monoclonal and polyclonal antisera. In most cases investigated viral antigens (human papilloma virus antigens or herpes simplex virus type 1 antigens) could be demonstrated in keratinocytic nuclei. Except for a few viral lesions in which epidermal Langerhans cells were rather numerous, epidermal Langerhans cells were reduced in number or absent in almost all viral lesions. Moreover, epidermal Langerhans cells and dermal dendritic cells showed changes in morphology, distribution, and immunophenotype. These alterations may be caused by a toxic effect of the virus on dendritic cells. HLA-DR+ keratinocytes could be identified in few viral lesions only; HLA-DQ+ keratinocytes were not seen. Possible explanations for this lack of MHC class II expression by keratinocytes are discussed.

Expression of HLA-DR and T6 antigens on keratinocytes and dendritic cells. A comparative immunohistochemical study.

The number, morphology, and distribution of cells with dendritic processes in the epidermis and dermis, as well as the expression of HLA-DR and T6 antigen on keratinocytes in 66 skin biopsy specimens have been studied. In the epidermis, OKT6+ cells with slender dendritic processes predominated in the upper layers and outnumbered OKIa1+ cells with dendritic processes, which were only fragmentarily stained and present throughout all layers. In the dermis, OKIa1+ cells with dendritic processes outnumbered OKT6+ cells with dendritic processes. Cases showing keratinocytic positivity for OKIa1 contained very few epidermal OKIa1+ cells with dendritic processes; cases showing surface staining of keratinocytes with OKT6 contained high numbers of epidermal OKT6+ cells with dendritic processes. We suggest that OKT6 and OKIa1 label distinctive subpopulations of epidermal and dermal dendritic cells and, as such provide complementary data.

Lymphocytes and dendritic cells in the normal uterine cervix. An immunohistochemical study.

OBJECTIVE: Assessment of the appearance, distribution and numerical density of immune cell populations in the normal human uterine cervix. SETTING: University Hospital Gasthuisberg. SUBJECTS: 29 healthy women undergoing total hysterectomy for non-cervical benign uterine disease. ANALYSIS: Immunohistochemistry and morphometrical analysis on histological sections containing ectocervix, transformation zone and endocervix, using antibodies against the following antigens: HLA-DR, CD4, CD22, CD1a and CD8. STATISTICAL ANALYSIS: Wilcoxon rank sum test. RESULTS: Lymphocytes in the epithelial and stromal compartments are predominantly T-lymphocytes. Intraepithelial T-lymphocyte and Langerhans' cell densities and their distribution are not influenced by the menstrual cycle and are the same in both ectocervix and transformation zone. CONCLUSION: The wide variation of T lymphocyte subpopulations and Langerhans' cell densities in the normal epithelium of the uterine cervix is stressed. We are the first to present a large and well-defined control series, which is indispensable to study the effect of smoking and other factors on the cervical immune system.

High grade squamous intraepithelial lesion (CIN 3) with extension into the endocervical clefts. Difficulty of cytologic differentiation from adenocarcinoma in situ.

OBJECTIVE: To elucidate the difficulty of cytologically differentiating high grade squamous intraepithelial lesion (SIL) with extension into the endocervical clefts from adenocarcinoma in situ (AIS). Criteria for the cytologic diagnosis of AIS have been delineated. However, it may sometimes be difficult to differentiate between AIS and carcinoma in situ (CIS). STUDY DESIGN: We reviewed cervical smears initially diagnosed as glandular intraepithelial neoplasia (GIN) with or without associated SIL CIN; in total, slides from 10 patients were studied. The final diagnosis in two cases was SIL plus GIN, in three cases high grade SIL (HSIL) (CIN 3) plus tubal metaplasia and in five cases HSIL without GIN. RESULTS: The cervical smears from the last five cases showed, besides features diagnostic of HSIL, the presence of large, crowded sheets with feathering, consisting of fusiform cells with an oval, bare, hyperchromatic nucleus, reminiscent of AIS. At one end of these sheets, normal endocervical cells were sometimes present. In all these cases the cone biopsy revealed HSIL with extension into the endocervical clefts. CONCLUSION: In retrospect, differential diagnosis with AIS is possible in most cases if diagnostic criteria are strictly applied. Indeed, contrary to AIS, feathering is often restricted to one end of the crowded sheets. Moreover, none of these crowded sheets contains glandular openings, and strips and rosettes with pseudostratification are absent.

Triple negative breast cancer: a study from the point of view of basal CK5/6 and HER-1.

AIM: Basal-like breast tumours, as defined by microarrays, carry a poor prognosis and therapeutic options are limited to date. Often, these tumours are defined as oestrogen receptor (ER) negative/progesterone receptor (PR) negative/human epidermal growth factor receptor 2 (HER-2) negative (triple negative) by immunohistochemistry (IHC), but a more complete definition should include expression of basal cytokeratins (CK5/6, CK14 or CK17) and/or human epidermal growth factor receptor 1 (HER-1). The aim of this study was to investigate to what extent CK5/6 and HER-1 characterise the group of triple negative breast cancers. METHODS: Expression of CK5/6 and HER-1 was studied by IHC in 25 triple negative breast carcinomas and 32 grade-matched, non-triple-negative controls. All 57 cases were further subjected to fluorescence in situ hybridisation to investigate HER-1 gene copy number. RESULTS: CK5/6 and HER-1 expression was most frequent in triple negative tumours: 22 out of 25 cases (88.0%) expressed at least one of these markers (60.0% CK5/6 positive and 52.0% HER-1 positive). In the control group, CK5/6 and HER-1 expression was found in ER-negative but not in ER-positive tumours (ER negative/PR negative/HER-2 positive tumours: 20.0% CK5/6 positive and 46.7% HER-1 positive). HER-1 gene amplification was found in five cases only: four triple negative (16.0%) and one ER-negative control (ER negative/PR negative/HER-2 positive, 6.7%). Of interest, all five HER-1 amplified cases showed a remarkably homogeneous HER-1 expression pattern. CONCLUSION: Expression of CK5/6 and HER-1 is frequent in ER-negative breast cancers, in triple negative and in non-triple negative tumours. In a minority of cases, HER-1 overexpression may be caused by HER-1 gene amplification. Further studies are needed to investigate whether such cases might benefit from anti-HER-1 therapy.