Maria Drijkoningen

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.

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.

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 χ2 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.

Polysomy 17 in Breast Cancer: Clinicopathologic Significance and Impact on HER-2 Testing

PURPOSE Polysomy 17 is frequently found in breast cancer and may complicate the interpretation of HER-2 testing results. We investigated the impact of polysomy 17 on HER-2 testing and studied its clinicopathologic significance in relation to HER2 gene amplification. PATIENTS AND METHODS In 226 patients with primary invasive breast carcinoma, HER2 gene and chromosome 17 copy numbers were determined by dual-color fluorescent in situ hybridization (FISH). The interpretation of FISH results was based on either absolute HER2 gene copy number or the ratio HER2/chromosome 17. Results were correlated with HER-2 protein expression on immunohistochemistry (IHC), HER2 mRNA expression by reverse transcriptase polymerase chain reaction (RT-PCR), and with various clinicopathologic parameters. RESULTS All cases with an equivocal HER-2 result by FISH, either by absolute HER2 copy number (44 of 226 patients; 19.5%) or by the ratio HER2/chromosome 17 (three of 226 patients; 1.3%), displayed polysomy 17. On its own, polysomy 17 was not associated with HER-2 overexpression on IHC or increased HER2 mRNA levels by RT-PCR. Moreover, and in contrast with HER2 gene amplification, polysomy 17 was not associated with high tumor grade, hormone receptor negativity, or reduced disease-free survival. CONCLUSION Polysomy 17 affects HER-2 testing in breast cancer and is a major cause of equivocal results by FISH. We show that tumors displaying polysomy 17 in the absence of HER2 gene amplification resemble more HER-2-negative than HER-2-positive tumors. These findings highlight the need for clinical trials to investigative whether polysomy 17 tumors benefit from HER-2-targeted therapy.

The E2F-regulated gene Chk1 is highly expressed in triple-negative estrogen receptor-/progesterone receptor-/HER-2-breast carcinomas

We previously showed that checkpoint kinase 1 (Chk1) and Claspin, two DNA-damage checkpoint proteins, were down-regulated by 1,25-dihydroxyvitamin D(3), a known inhibitor of cell proliferation. In the present study, we aimed to investigate the transcriptional regulation of Chk1 and Claspin and to study their expression levels in human breast cancer tissue. Transient transfection experiments in MCF-7 breast cancer cells showed that promoter activities of Chk1 and Claspin were regulated by the E2F family of transcription factors. Subsequently, transcript levels of Chk1, Claspin, and E2F1 were determined by quantitative reverse transcriptase-PCR analysis in 103 primary invasive breast carcinomas and were compared with several clinicopathologic variables in breast cancer. A strong correlation was found between Chk1 and Claspin transcript levels. Transcript levels of Chk1, Claspin, and E2F1 were highest in histologic grade 3 tumors and in tumors in which the expression of estrogen receptor (ER) and progesterone receptor (PR) was lost. Moreover, Chk1 expression was significantly elevated in grade 3 breast carcinomas showing a triple-negative ER-/PR-/HER-2- phenotype compared with other grade 3 tumors. Further research is warranted to validate the use of Chk1 inhibitors in triple-negative breast carcinomas for which treatment strategies are limited at present.

Tobacco smoking impairs the local immunosurveillance in the uterine cervix. An immunohistochemical study.

Previous reports have supported an association between tobacco smoking and cervical neoplasia. Our observations show an association between smoking and a reduction of the numerical densities of Langerhans cells and of helper/inducer T lymphocytes in the squamous epithelia of the transformation zone of the uterine cervix. This suggests a local impairment of cell-mediated immunity by smoking. This immunosuppressive effect could support the concept that smoking is an independent risk factor for cervical neoplasia.

Causes of inconsistency in diagnosing and classifying intraductal proliferations of the breast

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.

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.

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).

The complexity of genotypic alterations underlying HER2-positive breast cancer: an explanation for its clinical heterogeneity.

Purpose of review We discuss recent findings on the genotypic alterations associated with HER2-positive breast cancer in an attempt to clarify the clinical heterogeneity observed among these tumors. Recent findings Molecular genetic analysis supports the distinctive nature of HER2-positive breast cancer, which is primarily driven by HER2 gene amplification. Depending on the amplicon size, a variety of genes can be coamplified and overexpressed together with HER2, some of which may contribute to tumorigenesis; the amplicon size may even predict response to trastuzumab therapy. HER2 gene amplification may further destabilize the tumor genome, facilitating the generation of additional genomic aberrations including aneuploidy. The latter might imply polysomy 17, a phenomenon that should be discriminated from true HER2 gene amplification: polysomy 17 in the absence of HER2 gene amplification is not associated with HER2 overexpression nor with the clinical characteristics of HER2-positive breast cancer. Summary HER2 gene amplification is a complex event: it includes coamplification of other, potentially oncogenic genes and facilitates the generation of additional genomic aberrations. Further studies on these genotypic findings will be helpful to better identify the patients that might benefit from trastuzumab therapy.