Consistency in recognizing microinvasion in breast carcinomas is improved by immunohistochemistry for myoepithelial markers.

Microinvasion is the smallest morphologically identifiable stage of invasion. Its presence and distinction from in situ carcinoma may have therapeutic implications, and clinical staging also requires the recognition of this phenomenon. Microinvasion is established on the basis of several morphological criteria, which may be difficult and not perfectly reproducible among pathologists. The aim of this study was to assess the consistency of diagnosing microinvasion in the breast on traditional haematoxylin and eosin (HE) stained slides and to evaluate whether immunohistochemistry (IHC) for myoepithelial markers could improve this. Digital images were generated from representative areas of 50 cases stained with HE and IHC for myoepithelial markers. Cases were specifically selected from the spectrum of in situ to microinvasive cancers. Twenty-eight dedicated breast pathologists assessed these cases at different magnifications through a web-based platform in two rounds: first HE only and after a washout period by both HE and IHC. Consistency in the recognition of microinvasion significantly improved with the use of IHC. Concordance rates increased from 0.85 to 0.96, kappa from 0.5 to 0.85, the number of cases with 100% agreement rose from 9/50 to 25/50 with IHC and the certainty of diagnosis also increased. The use of IHC markedly improves the consistency of identifying microinvasion. This corroborates previous recommendations to use IHC for myoepithelial markers to clarify cases where uncertainty exists about the presence of microinvasion. Microinvasive carcinoma is a rare entity, and seeking a second opinion may avoid overdiagnosis.

FB1, a monoclonal antibody reacting with a keratin 14 epitope, stains only a small subset of psoriatic basal keratinocytes.

A murine monoclonal antibody, FB1, reacted with the basal keratinocytes of human stratified epithelia. One-dimensional and two-dimensional immunoblotting assays, performed on keratins extracted from HaCat cells and normal human keratinocytes, showed that FB1 recognizes K14. When LL002, another K14 monoclonal antibody is added, the FB1 stained area in the 2D-immunoblot seems to cover a fraction of the LL002 spot. Immunohistochemical data obtained from studies on normal human tissues supported the K14 specificity of FB1, but when compared with two other monoclonal antibodies, LL002 and RCK107 reacting with K14, some differences appeared. These differences were mainly seen in sweat glands, hair follicles, psoriatic epidermis and salivary glands. In psoriatic epidermis, FB1 showed a heterogeneous pattern of staining of the basal cell compartment. Intense reactivity was only observed at the bottom of the rete ridges. Staining diminished and finally disappeared in the basal cells above the dermal papillae. This observation supports the view that an increased germinative cell population in psoriasis involves a partially differentiated amplifying compartment in which the number of cell divisions is increased.

Epidermal growth factor receptors in human breast cancer: a plea for standardisation of assay methodology.

In a prospective study 200 primary human breast cancer specimens were analysed for epidermal growth factor receptor (EGFR) content by means of a multiple point ligand binding assay, proposed by the EORTC Receptor Study Group to be the standard EGFR assay. In 54% of the tumours the presence of saturable high affinity binding sites for epidermal growth factor could be demonstrated. The median EGFR level was 34 fmol/mg of membrane protein, the median Kd 0.50 nmd. Univariate analysis of the EGFR data stratified according to patient age, menopausal status, tumour size, axillary lymph node status, histological tumour type, tumour differentiation grade or the tumours' steroid hormone receptor status showed EGFR to be positively associated with younger age (P = 0.03), tumour dedifferentiation (P = 0.04) and steroid hormone receptor negativity (P less than 0.001). No association between EGFR and menopausal status, tumour size, axillary lymphnode status or histological tumour type could be demonstrated.

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.

Lethal osteopetrosis with multiple fractures in utero.

Severe osteopetrosis was diagnosed in utero in two successive pregnancies resulting from an intermarriage. Hydrocephaly and skeletal hyperdensity were detected at 18 weeks of gestation, and fractures at 24 weeks. We report on extensive ultrasound, radiological, and pathological findings, including those on brain and bone. The markedly reduced number of osteoclasts observed in these sibs and the very early fetal involvement suggest that this form of osteopetrosis might represent a new entity: autosomal recessive lethal osteopetrosis.

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.

Consistency achieved by 23 European pathologists from 12 countries in diagnosing breast disease and reporting prognostic features of carcinomas. European Commission Working Group on Breast Screening Pathology.

A detailed analysis of the consistency with which pathologists from 12 different European countries diagnose and classify breast disease was undertaken as part of the quality assurance programme of the European Breast Screening Pilot Network funded by the Europe against Cancer Programme. Altogether 107 cases were examined by 23 pathologists in 4 rounds. Kappa statistics for major diagnostic categories were: benign (not otherwise specified) 0.74, atypical ductal hyperplasia (ADH) 0.27, ductal carcinoma in situ (DCIS) 0.87 and invasive carcinoma 0.94. ADH was the majority diagnosis in only 2 cases but was diagnosed by at least 2 participants in another 14, in 9 of which the majority diagnosis was benign (explaining the relatively low kappa for this category). DCIS in 4 (all low nuclear grade) and invasive carcinoma (a solitary 1-mm focus) in 1. The histological features of these cases were extremely variable; although one feature that nearly all shared was the presence of cells with small, uniform, hyperchromatic nuclei and a high nucleo-cytoplasmic ratio. The majority diagnosis was DCIS in 33 cases; kappa for classifying by nuclear grade was 0.38 using three categories and 0.46 when only two (high and other) were used. When ADH was included with low nuclear grade DCIS there was only a slight improvement in kappa. Size measurement of DCIS was less consistent than that of invasive carcinoma. The majority diagnosis was invasive carcinoma in 57 cases, the size of the majority being 100% in 49. The remainder were either special subtypes (adenoid cystic, tubular, colloid, secretory, ductal/medullary) or possible microinvasive carcinomas. Subtyping was most consistent for mucinous (kappa, 0.92) and least consistent for medullary carcinomas (kappa, 0.56). Consistency of grading using the Nottingham method was moderate (kappa=0.53) and consistency of diagnosing vascular invasion, fair (kappa=0.38). There was no tendency for consistency to improve from one round to the next, suggesting that further improvements are unlikely without changes in guidelines or methodology.

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.

MR findings of a primary intramedullary malignant melanoma: case report and literature review.

A case of primary malignant melanoma of the conus medullaris depicted at MR imaging is presented. Tumoral histoimmunologic analysis revealed features of malignant melanoma. Because findings for primary melanoma outside the spinal cord were negative, the diagnosis of primary intramedullary malignant melanoma was established. This rare tumor should be suspected when T1-weighted images show signal hyperintensity and T2-weighted images show signal iso- or hypointensity, with mild contrast enhancement of the lesion. However, these features may vary depending on intratumoral bleeding and melanin content.

Locoregional recurrences after 649 modified radical mastectomies: incidence and significance.

A continuous series of 649 patients, treated by modified radical mastectomy for primary breast carcinoma, is analyzed after a median follow-up of 92 months. 'True isolated' locoregional recurrences (LR), defined as LR not preceded or followed by distant metastases within 6 weeks, appeared with a cumulative actuarial incidence rate of 6%, 14% and 19% after 1, 5 and 7 years respectively, whereas the respective figures for distant metastases (M1) were 10%, 37% and 48%. The main initial parameters, predicting both the LR-free and the M1-free interval, are presented by statistical analyses in the following order of importance: number of invaded lymph nodes in the axilla, tumor size (T) and histological grading of differentiation. The same factors also predicted the imminence of M1 once LR had occurred, as well as survival after LR. A higher incidence of M1 after LR was also correlated with estrogen-receptor negative tumors and with those LR occurring within one year after mastectomy. LR occurred at the chest wall (65%), in the sub-clavicular fossa (16%) and the axilla (6%); the remaining 13% occurred in two of the sites. There was a trend towards longer survival after chest wall recurrence than after LR recurrence at another site. Axillothoracic irradiations postmastectomy gave a lower rate of LR in 227 patients than did a regimen of 12 months adjuvant chemotherapy with irradiation restricted to the internal mammary lymph nodes in 120 subsequent patients: 17 vs 25% at 5 years (P = 0.03 when adjusted by initial nodal involvement and T-size). Total excision of LR (repeated if new LR occurred) gave better rates of local ultimate control and survival than other kinds of treatments, with or without adjuvant local or systemic therapy. LR is not always a sign of imminent generalized disease. Actuarial 5-year survival after LR is 26.2% overall whereas, if only 'true isolated' LR are considered, the survival is 37%.

Primary carcinoid tumors of the middle ear. Report on four cases and a review of the literature.

Carcinoid tumors are rare in the middle ear. To our knowledge, only 17 cases could be found in the literature, the first of which was described in 1980. In addition to enlarging on a previous observation we present three new cases. The neoplasms showed a striking, heterogeneous aspect ranging from solid trabecular to tubuloglandular growth patterns resembling the classic carcinoid tumor and adenomatous middle ear tumor, respectively. Based on immunohistochemistry and electron microscopy, three cell types were found. A review was made of our four patients and the cases described in the literature. The medical histories ranged from 1 month to 9 years. Presenting symptoms and signs were not characteristics, but hearing loss predominated. In two patients, the eardrums were perforated, in all the others it was intact and often bulging. Surgery, usually radical mastoidectomy, was performed in all cases. Often the tumor encased the ossicular chain, without infiltration. In two patients, local recurrence occurred that was treated successfully with surgery. All the cases showed an indolent biological course and the tumors were clinically nonfunctional, despite the recognition of biogenically active products by immunohistochemistry. To our knowledge, regional or distant metastases have never been reported. Conservative surgery with radical removal of the primary or recurrent tumor is the treatment of choice.

Adeno-carcinoid or amphicrine tumors of the middle ear a new entity?

The clinicopathological, ultrastructural and immunohistochemical characteristics of four primary tumors of the middle ear are reported. These neoplasms showed a striking, heterogeneous aspect ranging from solid-trabecular (Type I) to tubulo-glandular (Type II) growth patterns. Secretory activity of the tumor cells was evaluated by immunohistochemistry and electron microscopy. Based on these procedures, three cell types were found, mainly limited to tumors with a tubulo-glandular (Type II) growth pattern. Most frequent were B-cells with an abundant pale cytoplasm containing neuroendocrine granules, both cytokeratin and vimentin as well as several endocrine marker substances. Less frequent were A-cells, which are slender, darkly staining and line the glandular lumina. They showed exocrine activity only and stained strongly with a polyclonal cytokeratin antibody. Finally, least frequent were amphicrine cells, which were characterized by both lumina and neuroendocrine granules in their cytoplasm and were interpreted as the link between A and B cells. Although this morphological description closely resembles that of carcinoids and adenocarcinoids of the respiratory tract and gut, the clinical behaviour of these middle ear tumors nevertheless seems different, with no recurrence or metastasis after a follow-up of 1 to 14 years (median 78 months). Therefore, some authors suggest that these tumors should be classified as middle ear adenomas or adenomatous tumors. However, we strongly feel that these tumours represent a distinct entity and can be classified as adenocarcinoids or amphicrine tumors, i.e. demonstrating both exocrine and endocrine activities. Further work is required to evaluate the exact proportion of neuroendocrine and amphicrine tumors in the heterogeneous group of adenomas and in the rarely described group of adenocarcinomas.

Consequences of a National Mammography Screening Program on diagnostic procedures and tumor sizes in breast cancer. A retrospective study of 1540 cases diagnosed and histologically confirmed between 1995 and 1997.

In 1992, a national screening mammography program, including female patients between 50 and 64 years of age, was launched in Luxembourg. The effects of this campaign on the different diagnostic procedures, especially fine needle aspirations (FNA), large core needle biopsies (LCNB), and surgical specimens, were analyzed. From 1983 to 1997, the National Cancer Registry recorded 3167 new cases of invasive female breast cancer, all histologically diagnosed in one central pathology department. In 1996, the population consisted of 418,300 inhabitants (212,900 females). The number of breast cancer, tumor size, the nature of the diagnostic procedures, their diagnostic value as well as the number of physicians, "aspirators", and "biopsists" were evaluated. Between 1992 and 1994, the incidence of invasive breast cancers increased, concomitant with the launching of a National Screening Mammography Program. The diagnosis of in situ cancers tripled, and the mean size of invasive breast cancer decreased from 2.1-2.4 cm to 1.1-1.4 cm. Since 1994, the number of FNA had remained stable, LCNB had increased by 417.5%, and surgical biopsies had decreased by 18.95%. Between 1995 and 1997, 28.37% of 1075 FNA, and only 9.6% of 465 LCNB yielded inadequate samples. FNA were done by 77 different doctors (53.25% being gynecologists) and LCNB by 34 (52.94% being radiologists). The first diagnoses of all invasive cancers (n = 790) were made by using frozen sections from surgical specimens in 58.35% (n = 461), LCNB in 18.23% (n = 144), mastectomy in 10.13% (n = 80), formalin-fixed biopsies in 9.49% (n = 75), and FNA in 3.17% (n = 25). There are beneficial effects (increase in the number of diagnoses of in situ cancer; decrease in tumor sizes) not only for the "target" age group (50-64 years), but also for all female age groups (> 15 years). For quality assurance purposes, it is absolutely recommended to carry out pathological, radiological, and diagnostic work in specialized centers.

Three dimensional imaging of mammary ductal carcinoma in situ: clinical implications.

The conservation treatment of ductal carcinoma in situ (DCIS) is based on the surgical excision of the tumour together with irradiation of the remaining breast. Because short-term recurrence is almost certainly caused by residual tumour, an attempt should be made to verify the adequacy of the excision by assessing the specimen margin. The reliability of histologic margin assessment is influenced by the growth pattern of DCIS within the ductal tree and by the distance between tumour foci. Using an original stereoscopic technique, the present study of 60 mastectomy specimens shows that continuous and multifocal growth patterns are usual. A multifocal distribution (defined as gap of 4 cm or more between tumour foci) was found in only a single case. The growth pattern is related to DCIS type. Poorly-differentiated DCIS shows continuous growth, in contrast to the well-differentiated DCIS, which has a multicentric distribution. Irrespective of histologic type, however, only 8% of DCIS have a multifocal distribution with gaps greater than 10 mm. Therefore, with careful assessment, the likelihood of a false free margin seems theoretically low and should encourage the use of conserving treatment for eradicable DCIS.

Ductal carcinoma in situ: a proposal for a new classification.

Details of a proposed new classification for ductal carcinoma in situ (DCIS) are presented. This is based, primarily, on cytonuclear differentiation and, secondarily, on architectural differentiation (cellular polarisation). Three categories are defined. First is poorly differentiated DCIS composed of cells with very pleomorphic, irregularly spaced nuclei, with coarse, clumped chromatin, prominent nucleoli, and frequent mitoses. Architectural differentiation is absent or minimal. The growth pattern is solid or pseudo-cribriform and -micropapillary (without cellular polarisation). Necrosis is usually present. Calcification, when present, is amorphous. Second, at the other end of the spectrum is well-differentiated DCIS, composed of cells with monomorphic, regularly spaced nuclei containing fine chromatin, inconspicuous nucleoli, and few mitoses. The cells show pronounced polarisation with orientation of their apical border towards intercellular spaces usually resulting in cribriform, micropapillary and clinging patterns, although a solid pattern of well-differentiated DCIS also occurs. Necrosis is uncommon. Calcifications, when present, are usually psammomatous. The third category, intermediately differentiated DCIS, is composed of cells showing some pleomorphism but not so marked as in the poorly differentiated group. There is, however, always evidence of polarization around intercellular spaces, although this is not so pronounced as in the well-differentiated group. These two criteria, cytonuclear differentiation and architectural differentiation, have been found to be more consistent throughout a DCIS lesion than previously employed criteria of architectural pattern or the presence or absence of necrosis.

Identification of Campylobacter pylori in gastric biopsy smears.

The use of gastric biopsy imprint smears to diagnose Campylobacter pylori was compared with the use of tissue sections and cultures. Multiple gastric biopsies were taken from the mucosa of 42 patients during endoscopy. Imprint smears were prepared from the samples used to make tissue sections; other samples were used for microbiologic culture. There was a good concordance (93%) between the morphologic diagnosis of C pylori in the air-dried, Giemsa-stained smears and the tissue sections; the cytologic preparations were clearly positive in six cases (14%) whose sections contained low numbers of the organisms. There was a concordance of 83% between the combined morphologic techniques and the bacteriologic culture. Six positive cases were detected only by the morphologic techniques while one positive case was detected only by bacteriologic culture. C pylori was identified in one or more preparations of the antral biopsy specimens in 23 (55%) of the 42 cases, including 23 (74%) of the 31 cases with a final diagnosis of gastritis or ulcer. These results show the usefulness of the cytologic study of gastric biopsy smears in diagnosing C pylori infections.

Comparison of biochemical and immunoenzymatic macromethods and a new immunocytochemical micromethod for assaying estrogen receptors in human breast carcinomas.

Estrogen receptors (ERs) were assayed in 23 breast carcinomas by: (1) the conventional biochemical assay with dextran-coated charcoal (DCC); (2) the immunoenzymatic assay using a monoclonal antibody (MAb), ER-EIA (Abbott); and (3) an original cytochemical method using another MAb, ER-ICA (Abbott). The first two techniques were performed on biopsy samples, whereas the last was carried out on fine needle aspiration (FNA) samples. The ER contents in aspirates were evaluated by: (1) scaled proportions of colored neoplastic cells; (2) scaled coloration intensity; (3) total grading (= proportion plus intensity); (4) product grading (= proportion times intensity); and (5) a new index (NI) described in this paper. The ER-EIA assay correlated best, with a high statistical significance, with the NI (P less than .001); NI was also the only index that significantly correlated (P less than .05) with the DCC results. The results show that the ER-ICA assay offers the great advantages of being applicable to FNA specimens and of producing rapidly available results. This new technique enriches the panel of MAbs for the diagnosis of adenocarcinomas and offers a new tool for the therapeutic follow-up of breast cancer patients. Our preliminary results suggest that the anti-ER MAbs might be helpful for measuring the hormone dependence of small lesions not assayable by DCC, even under endocrine therapy, thus avoiding false-negative assays.