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.

Belgian consensus guidelines for follow-up of women with cervical cytological abnormalities.

In medical care cervical cancer screening is important because it enables the detection of precancer and cancer at an early stage. By adequate treatment after a screening-detected lesion it helps to reduce the mortality related to cervical cancer. Worldwide, many millions of women have smears taken at a more or less regular base and of these, approximately 7% are abnormal, and follow-up is thus required.As this represents an important cost in medical health care and has serious consequences for the affected women, it is important to have uniform and clear guidelines to allow an optimal follow-up and clinical management. A system for the uniform reporting of cervical cytology has been designed by the National Cancer Institute (U.S.A.) and resulted in the Bethesda System 1991. The present paper and the terminology used are based on the Bethesda System revised in 2001. It explains the guidelines, based on the 2001 Bethesda System and the 2004 consensus guidelines for the management of women with cervical cytological abnormalities, as developed by the members of the Board of the Belgian Society of Clinical Cytology, and adapted to the Belgian situation.

Expression of the BRCA1-interacting protein Brip1/BACH1/FANCJ is driven by E2F and correlates with human breast cancer malignancy.

Mutations in the BRCA1-interacting DEAH helicase Brip1 confer an increased risk of breast cancer. In the present study we aimed to unravel the transcriptional control of Brip1 and to determine its expression levels in a set of 101 primary invasive breast carcinomas. Transcription of Brip1 was found to be cell growth-related and controlled by the E2F/retinoblastoma (Rb) pathway through a conserved E2F-responsive site. Repression of Brip1 expression by the cell growth-inhibiting compound 1alpha,25-dihydroxyvitamin D3 depended on this same E2F-responsive site. In spite of its role as a tumor suppressor, both quantitative reverse transcriptase-PCR analyses and immunohistochemical stainings showed significantly elevated Brip1 expression levels in grade 3 tumors as compared to grade 1 or 2 carcinomas. Furthermore, increased Brip1 transcript levels were found in tumors with an estrogen receptor-negative, progesterone receptor-negative or HER-2-positive status. In conclusion, these data show that Brip1 is a genuine target gene for the E2F/Rb pathway and that elevated expression levels of Brip1 are detected in primary invasive breast carcinomas with unfavorable characteristics.

Age interacts with the expression of steroid and HER-2 receptors in operable invasive breast cancer.

BACKGROUND: The negative association between the oestrogen receptor (ER) and the human epidermal growth factor receptor 2 (HER-2) in breast cancer travels in both directions. ER+ tumours are less likely HER-2+ and HER-2+ tumours are less likely ER+. METHODS: We studied the age-related immunohistochemical (IHC) expression of ER, progesterone receptor (PR) and HER-2 in 2,227 tumours using age as a continuous variable. Steroid receptors were considered positive for any nuclear staining of invasive cancer cells and for HER-2, either for strong expression by IHC (score 3+) or gene amplification by fluorescence in situ hybridisation (FISH). Based on nonparametric regression, the age-related association between steroid receptors and HER-2 was presented as likelihood curves. RESULTS: The association between ER or PR and HER-2 is age-related. The age-related expression of ER and PR is HER-2 dependent. In HER-2(-) cases, the odds ratio (OR) for being ER+ was 2.594 (95% CI = 1.874-3.591) up to age 50 and age-independent thereafter; for PR-expression the OR was 2.687 (95% CI = 1.780-4.057) up to age 45 and 0.847 (95% CI = 0.761-0.942) thereafter. In HER-2+ cases, the OR was 0.806 (95% CI = 0.656-0.991) to be ER+ and 0.722 (95% CI = 0.589-0.886) to be PR+. The age-related OR for breast cancers to be HER-2+ is steroid receptor dependent. Taking together, ER+PR+HER-2+ breast cancers appear on average 5.4 years earlier than breast cancers of any other ER/PR/HER-2 phenotype (95% CI = 3.3-7.5; P < 0.0001). CONCLUSION: There is a qualitative interaction between age and expression of steroid and HER-2 receptors. Our findings suggest a strong age-related selective growth advantage for breast tumour cells belonging to the ER+PR+HER-2+ subgroup.

The future of cytopathology in Europe. Will the wider use of HPV testing have an impact on the provision of cervical screening?

The emphasis of the EFCS Congress held in Venice in October 2006 was on the future of Cytopathology in relation to events in Europe. Much of the discussion centred on the role of human papilloma virus testing and its impact on the provision of cervical screening. The following is a transcript of the discussion that took place at the Advisory Board Meeting for the journal Cytopathology, with some additional written comments received prior to the meeting. A brief summary has been provided as a conclusion by Dr A. Herbert.

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.

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.

Prediction of recurrence after treatment for high-grade cervical intraepithelial neoplasia: the role of human papillomavirus testing and age at conisation.

OBJECTIVES: The aim of this study was to examine the accuracy of the presence of high-risk human papillomavirus (HR-HPV) DNA (HR-HPV DNA test) postconisation as prediction of recurrent or residual cervical intraepithelial neoplasia (CIN) after treatment of high-grade cervical intraepithelial lesions (CIN2+) in a prospective study and to compare this with follow-up cytology and the marginal status of the excised tissue. DESIGN: Prospective follow-up study. SETTING: Unselected women presenting at colposcopy clinic of University Hospital Gasthuisberg, Leuven. POPULATION: Seventy-two women treated with conisation for CIN2 or CIN3. METHODS: Women were followed by HR-HPV DNA test (Hybrid Capture II test of Digene) every 3 to 6 months. The same vial was used for cytology and the HR-HPV DNA test (SurePath). All women were further followed by colposcopy and cytology for 24 months at 6-month intervals. The outcome of the study was presence of >CIN2, proven with colposcopy-directed biopsy occurring within 24 months after treatment. HR-HPV status was correlated with recurrent or residual CIN2+. MAIN OUTCOME MEASURES: Sensitivity, specificity, predictive values and diagnostic odds ratios to predict treatment failure or cure were computed for HR-HPV testing, marginal status and follow-up cytology. HR-HPV status was also correlated with section margins postconisation and with the first cervical smear. RESULTS: In 6 of the 72 treated women (8%), residual or recurrent CIN occurred. Women with recurrence were significantly older than women without a recurrence (51.5 +/- 9.6 versus 39.8 +/- 12.2 years, P= 0.007). All six women with recurrence were HR-HPV positive, four had a positive follow-up smear (>or=atypical squamous cells of uncertain significance = ASCUS+) and only two had involved section margins. Among the 66 cured women, 15 were HR-HPV positive, 6 had an abnormal smear and 12 had positive section margins. Sensitivity of cytology, positive section margins and HR-HPV DNA positivity was 66.7, 33.3 and 100% to predict treatment failure. Specificity of the three tests was, respectively, 90.9, 81.8 and 77.3%. Women with HR-HPV DNA at 3 to 6 months showed recurrent or residual CIN in 15% (2/13) if they had normal follow-up Pap smears and in 50% (4/8) if they had abnormal Pap smears. Margin status was not statistically significantly associated with human papillomavirus status. CONCLUSION: Persistence or clearance of HR-HPV DNA is an early valid prognostic marker of failure or cure after treatment for CIN2+ and is more accurate than cytology or section margin status at the time of conisation. The absence of HR-HPV DNA has a 100% negative predictive value. Higher age at conisation may be a previously unrecognised risk factor for recurrence.

Amyloid deposition of the breast in primary Sjögren syndrome.

We report amyloid deposition in the breast presenting as suspicious microcalcifications on screening mammography. Stereotactic mammotome biopsy provided the diagnosis. The history of the patient revealed primary Sjögren syndrome. The combination of amyloid deposition in the breast and Sjögren's syndrome has only rarely been reported.

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.

Hormone receptors do not predict the HER2/neu status in all age groups of women with an operable breast cancer.

BACKGROUND: In breast cancer, there is an inverse relationship between HER2/neu overexpression and receptors for estrogen (ER) or progesterone (PR). Some clinical observations such as the age-related association between hormone receptors and tumour grade, which predicts HER2/neu overexpression, suggest an age-related relationship. PATIENTS AND METHODS: Our study population consisted of 1362 consecutive women receiving primary surgery for non-metastatic invasive breast cancer. We compared the relationship between both hormone receptors and HER2/neu overexpression in different age groups taking other tumour characteristics into account. RESULTS: In a multivariate model, considering the overall group, a negative ER, a negative PR and a high tumour grade were predictive for HER2/neu overexpression (P <0.001). Considering 246 women aged < or =45 years, the only predictor for HER2/neu overexpression in this age category was a high tumour grade (P = 0.003). Considering the 1116 women aged >45 years, ER (P = 0.001), PR (P = 0.001) and tumour grade (P <0.001) were associated with HER2/neu (P <0.001). CONCLUSION: Our findings indicate that the association between ER, PR and HER2/neu overexpression varies with age. The hormone receptors are not an independent predictor for the HER2/neu status in young women while they are in elder patients.

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.

Real-time reverse transcription-PCR and fluorescence in-situ hybridization are complementary to understand the mechanisms involved in HER-2/neu overexpression in human breast carcinomas.

AIMS: To evaluate the HER-2/neu status at the mRNA and DNA level of breast carcinomas and to compare it with HER-2/neu receptor overexpression by immunohistochemistry (IHC). METHODS AND RESULTS: In 32 invasive breast carcinomas, frozen tissue was available for real-time detection of HER-2/neu mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR). Corresponding paraffin sections were examined by IHC and fluorescence in-situ hybridization (FISH). Thereby, different IHC and FISH procedures were compared. Using microwave epitope retrieval, all 32 cases scored 3+ on IHC, whereas only 28 out of 32 cases scored IHC 3+ using water bath epitope retrieval. All of these 28 cases showed increased levels of HER-2/neu mRNA. Dual-colour FISH analysis showed corresponding gene amplification in all 28 cases, with two cases showing a peculiar amplification pattern. In the remaining four cases, scoring IHC 2+ using water bath epitope retrieval, mRNA levels were not elevated. Three cases did not have gene amplification and one case showed low-level HER-2/neu gene amplification. All four carcinomas showed chromosome 17 polysomy. CONCLUSIONS: Real-time RT-PCR is accurate in selecting breast carcinoma cases scoring 3+ by IHC with high-level gene amplification. Results obtained by dual-colour FISH suggest that mechanisms leading to HER-2/neu receptor overexpression may be different between carcinomas scoring 2+ and 3+ on IHC, with polysomy 17 found in the former and gene amplification in the latter.

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.

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.

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.

Effect of TNP-470 (AGM-1470) on the growth of rat rhabdomyosarcoma tumors of different sizes.

Potential anticancer therapy with the fumagillin analog TNP-470 was investigated in the present project using subcutaneously growing rhabdomyosarcomas in rats. Specifically, influences of different tumor sizes at the start of treatment as well as dose/schedules were evaluated with this angiogenesis inhibitor. The results show a significant (p = < or = 0.01) reduction of the growth rate, even for relatively large-sized (> 7 cm3) tumors, when 50 mg/kg TNP-470 was used every other day for up to 3 or 5 injections. With 30 mg/kg TNP-470 injections, effects were seen only with tumors measuring < 7 cm3. The histologic examinations demonstrate an increase in necrosis, both in the center and in the peripheral part of TNP-470-treated tumors. Overall, both tumor volume and drug dose determine treatment outcome with the rat rhabdomyosarcoma. The results suggest that angiogenesis inhibitors could represent a valid component in the treatment of progressive tumor growth, also of large tumors as often encountered in clinics. The antivasculature therapy might also improve hypoxia/necrosis-related therapeutic approaches.

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.