An ultrasonography-cytology protocol for the diagnostic management of regional nodes in a subset of patients with Merkel cell carcinoma of the skin.

BACKGROUND: The status of regional lymph nodes (LNs) is one of the most consistent predictors of survival in Merkel cell carcinoma (MCC). In cases of clinically localized disease, current practice involves sentinel lymph node (SLN) assessment. OBJECTIVES: To propose ultrasonography (US) followed by fine needle aspiration cytology (FNAC) and immunohistochemistry as a useful diagnostic tool in the pre-surgical management of patients with MCC. METHODS: US of LNs was performed in 75 patients with MCC (22 with stage III tumours; 53 with stage I-II). In patients with US suspected disease, US coupled with FNAC of the LN was performed. Smears were examined by routine cytological staining supplemented with immunohistochemical staining for cytokeratin 20. All patients underwent surgical removal of regional LNs. RESULTS: In all 22 patients with stage III tumours, US was indicative of tumour deposits and FNAC confirmed metastases to LNs. In 11 of 53 patients with localized MCC without clinical evidence of nodal disease, US revealed enlarged, equivocal nodes where FNAC was performed. Ten LNs were cytologically positive for metastases, and one was negative. Upon histological examination, the FNAC-negative case showed a metastasis 5 mm in diameter. In all the other 42 cases with no clinical or US evidence of LN involvement, only SLN biopsy was performed and in six cases small metastatic foci were detected. Ultimately, of the 53 stage I-II MCC, 17 had positive LN involvement. In 10 cases (59%) metastases were detected by FNAC, and in seven cases, were detected by SLN biopsy. CONCLUSIONS: In a selected subset (∼20%) of patients with MCC with clinically localized disease, US followed by FNAC in the suspect LN is a valid alternative to the classical protocol of SLN histological examination.

Biological components in a standardized derivative of bovine colostrum.

Products of different origin, time of collection, and activities fall under the general term of colostrum and, therefore, great variability in composition as well as in the concentration of its components has been reported in the literature. In the present study, we describe the standardization of a bovine colostrum derivative and the characterization of its bioactive components. Evaluation of the most representative agents (lactoferrin, transferrin, IL-2, IFN-γ, tumor necrosis factor, IgG, and IgA) showed that a marked decrease in active components occurs after the first few hours. Bovine colostrum was, therefore, collected up to the fifth hour after delivery from Holstein cows, in the presence of preservatives, and immediately frozen. A protocol of centrifugation, filtration, and lyophilization was then applied to pools of colostrum from at least 30 cows to obtain a stable, sterile, standardized product. Preservatives were removed by dialysis. Evaluation of the active biological components of colostrum showed that the final product of colostrums contained significant and reproducible amounts of bioactive factors, including cytokines, immunomodulating factors, growth factors, and immunoglobulins. The final product appeared, therefore, as a sterile, pyrogen-free, standardized derivative of bovine colostrum with a high concentration of bioactive components.

Diagnosis of deep-seated lymphomas by endoscopic ultrasound-guided fine needle aspiration combined with flow cytometry.

OBJECTIVE: Although endoscopic ultrasound combined with fine needle aspiration (EUS-FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS-guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. METHODS: Of 1560 patients having EUS-guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS-FNA. There was adequate material to perform FC analysis for all but one case. RESULTS: EUS-FNA-FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. CONCLUSIONS: Our results show that a combination of EUS-FNA-FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep-seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non-lymphoma cases.

Nuclear shape in papillary thyroid carcinoma: a role for lamin B receptor?

Irregularity in the nuclear shape, with extensive folds and invaginations of the nuclear membrane (NM), remain the basic diagnostic feature of papillary thyroid carcinoma (PTC). The biological reasons for these irregularities are obscure, but evidence has been presented that they might be linked to RET÷PTC gene translocation. In the present study, we have investigated the hypothesis that the NM irregularities in PTC might be linked to alterations in the expression of lamin B receptor (LBR), a component of the inner NM responsible for the distribution of Lamin B and associated chromatin. Fisher AH et al. already reported on the lack of LBR in PTC, a finding in contrast with the observation that a reduced expression of LBR because of gene mutation is responsible for the lack of nuclear segmentation of granulocytes in Pelger-Huët anomaly. In the present study, we confirmed the lack of immunohistochemical staining for LBR in PTC nuclei, in contrast to a positive staining in intestinal epithelium and stromal cells. However, Western blot and RT-PCR analysis demonstrated a strongly positive reaction in PTC extracts, thus proving an expression of LBR higher in PTC cases and cells than in follicular carcinoma cells. In conclusion, our data suggest that LBR is heavily expressed in PTC cells, but an abnormal folding of the protein might explain its lack of immunohistochemical reactivity and be associated with the anomalous folding of the NM.

Proper detection of the nuclear shape: ways and significance.

Shape and size of the nucleus, coupled with changes in chromatin amount and distribution, still remain the basic microscopic criteria for cytological diagnoses. Diagnostic recognition of the nuclear shape in pathological histology and cytology has been always based on the assumption that it is the content in nucleic acids, which determines the nuclear shape. The present review challenges this opinion, focuses on the structure, and functions of the nuclear envelope and on how these features can be exploited in diagnostic pathology. In particular, we will present the contribution of thee-dimensional modeling to the understanding of nuclear irregularities in breast cancer and papillary thyroid carcinomas. Specifically, it will be shown how tagging the nuclear membrane with anti-Emerin antibodies can represent an additional and valuable tool in the differential diagnosis of thyroid lesions. Finally, the prognostic importance of detecting irregularities of the nuclear shape in breast carcinomas by immunofluorescence staining for nuclear proteins will be discussed.

Safe transportation of formalin-fixed liquid-free pathology specimens.

Diagnostic pathology activities are largely based on fixation of tissues in 4% formaldehyde, which has recently been re-classified as a carcinogenic compound and banned in several countries. Hospitals that do not have in-house pathology services need to send surgical and biopsy specimens to referral centers. These are generally transferred in liquid containers, under suboptimal safety conditions, as accidental spillage of potentially dangerous substances may occur. A safe, innovative, two-step procedure for pathology sample transportation is presented. Formalin-fixed material from ten surgical cases was dissected (including surrogate biopsies) and preserved in liquid-free plastic bags under vacuum for up to 30 days and subsequently processed for conventional histology, several immunohistochemical markers, and molecular tests (e.g., RAS mutation). The data were compared with the corresponding routine analyses. Formalin-fixed specimens after up to 30 days under vacuum storage gave equivalent results compared to standard histopathological slides and molecular tests, regarding both hematoxylin-eosin, immuno-stained slides and also nucleic acid extracted for molecular tests. The proposal of under-vacuum sealing pathology specimens that were previously formalin fixed can be adopted to transfer liquid-free biopsy and surgical specimens to referral pathology services. In fact, it is easy to perform, less expensive (both plastic bags and domestic-type vacuum chamber machines are at affordable costs), and above all is fully safe and adequate in the pre-analytical processing of pathology specimens.

Patients with advanced stage breast carcinoma immunoreactive to biotinylated Herceptin are most likely to benefit from trastuzumab-based therapy: an hypothesis-generating study.

BACKGROUND: Biotin-labeled trastuzumab (BiotHER) can be used to test for HER2 by immunohistochemistry. We previously showed that BiotHER immunoreactivity is highly correlated with HER2 amplification and indicated that it could be associated with better clinical outcome in advanced breast cancer patients receiving trastuzumab. PATIENTS AND METHODS: Tumor specimens and clinical information from 234 patients who received trastuzumab-based treatments were collected from 10 institutions. HER2 amplification and BiotHER immunoreactivity were assessed centrally. The effect of BiotHER positivity on response rate (RR), time to progression and survival were studied by univariate and multivariate analysis in patients presenting HER2-amplified breast cancer. The pathologic reviews of the assays were blinded to patient outcomes. RESULTS: BiotHER was positive in 109/194 (56%) HER2-amplified breast cancers and in one not amplified tumor. RRs were 74% [95% (confidence interval) CI 64%-81%] and 47% (95% CI 36%-58%) in BiotHER-positive and -negative tumors, respectively (P < 0.001). BiotHER immunoreactivity was independently associated with increased probability of tumor response (odds ratio 3.848; 95% CI 1.952-7.582), with reduced risk of disease progression [hazard ratio (HR) 0.438; 95% CI 0.303-0.633] and with reduced risk of death (HR 0.566; 95% CI 0.368-0.870) by multivariate analysis. CONCLUSION: The results support a role for BiotHER testing in better tailoring trastuzumab-based treatments in patients with advanced HER2-amplified breast cancers.

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.

Endoscopic ultrasound-fine needle aspiration (EUS-FNA) for pancreatic lesions: effectiveness in clinical practice.

BACKGROUND: Diagnosis of pancreatic masses is often difficult. Endoscopic ultrasound-fine needle aspiration has been proposed as the best single-step strategy. AIMS: To prospectively evaluate feasibility, effectiveness and safety of endoscopic ultrasound-fine needle aspiration of pancreatic masses in a consecutive study of unselected patients. METHODS: Two hundred ninety-three patients were enrolled in two referral Hospitals in Northern Italy. All patients were referred either due to the presence of imaging test abnormalities (suspected or evident masses, or features indirectly suggesting the presence of a mass) or due to clinical or biochemical findings suggesting pancreatic cancer in the absence of positive imaging. All patients underwent linear array endoscopic ultrasound and, when indicated, fine needle aspiration. All procedures were recorded prospectively. The final diagnosis was established at the end of follow-up or when the patients underwent surgery or died. RESULTS: Fine needle aspiration was indicated in 246 of 293 cases (84%), considered technically feasible in 232 of 246 cases (94%) and gave adequate samples for histopathological diagnosis in 204 of 232 cases (88%). Endoscopic ultrasound sensitivity, specificity and accuracy were 79, 60 and 72%, respectively; the corresponding figures for endoscopic ultrasound-fine needle aspiration were 80, 86 and 82%. There was good agreement with final diagnosis for endoscopic ultrasound-fine needle aspiration (kappa 0.673, 95%CI 0.592-0.753), greater than that for endoscopic ultrasound alone (kappa 0.515, 95%CI 0.425-0.605). There was one case of intracystic haemorrhage and one case of transient hyperthermia (0.3%). CONCLUSIONS: Endoscopic ultrasound-fine needle aspiration of pancreatic masses seems to be feasible, effective and safe in this consecutive study of patients.

[Histological sectioning of brush bristles allows an improved diagnosis of biliary tract lesions]. / Il sezionamento istologico delle setole dello spazzolino permette di migliorare la diagnosi delle lesione delle vie biliari.

Biliary tract brush cytology is increasingly being recognized as a favoured method for evaluating abnormalities of the biliary tract. In order to increase the diagnostic accuracy, we devise a new brush processing method finalized to the complete and ideal cytologic examination of the collected material. Small fragments of the mucosa, of inflammatory cell aggregates or of carcinomas are observed and the results are optimally fixed and allow a definitive histological diagnosis.

[On-site evaluation and triage for endoscopic ultrasound-guided fine needle aspiration cytology. The Turin experience]. / Valutazione on-site e triage per la citologia agoaspirativa sotto guida ecoendoscopica. L'esperienza torinese.

AIM: Evaluation of the importance of the on-site presence of a skilled cytopathologist during endoscopic ultrasound-guided fine needle aspiration at determining samples' adequacy and performing ancillary techniques which can be helpful for the diagnosis. METHODS: A retrospective analysis of our institute's experience with EUS-FNA sampling is presented. From January 2001 to May 2007, 404 patients underwent the EUS-FNA evaluation. From 2003 a cytopathologist was present during the procedure and started making an extemporary evaluation of the samples' adequacy. RESULTS: Before 2003, a final cytological diagnosis was available in only 70% of the cases (without an on-site cytopathologist). After 2003, in 90% of the cases (with an on-site cytopathologist). It is possible planning and performing: immunocytochemistry on cell block material including evaluation of the proliferation index; to obtain a sample for the flow cytometry in cases of lymphomas or a microbiologic workup in cases of infective lesions. CONCLUSION: The quality of the specimens and the proper handling of the aspirated sample are very important to succesfully obtain a definitive cytological diagnosis in EUS-FNA. On-site evaluation and triage of the material allow to improve the accuracy of the diagnosis.

Production of casein and the presence of estrogen receptors in human breast cancer.

The relationship between the presence of estrogen receptors and casein (evaluated on a semiquantitative basis with a specific immunofluorescence method) was statistically analyzed in 50 cases of human breast carcinomas. No significant correlation was found between these two parameters, whereas a relationship was established between the production of casein and the degree of histological differentiation. The results of this study, like those of other studies, revealed a lack of correlation between the presence of estrogen receptors and the degree of histologic differentiation.

CAR-3, a monoclonal antibody-defined antigen expressed on human carcinomas.

Several monoclonal antibodies were raised against the human epidermoid carcinoma line A 431. The antibody produced by clone AR-3, when tested in enzyme-linked immunosorbent assay, was found to react with the cell line used as immunogen, the human gastric carcinoma line KATO III, the colon carcinoma line HT29, and the ovarian carcinoma line SW626. This monoclonal antibody was found unreactive when tested on human peripheral blood leukocytes or on a number of normal or neoplastic cell lines. The antibody precipitated a high-molecular-weight glycosylated component. When tested on paraffin sections by the avidin:biotin: peroxidase method, the AR-3 antibody stained pancreatic (6:7), gastric (11:14), ovarian (5:6), colon (4:8), endometrial (4:6), and cervical (4:7) carcinomas. A small minority of carcinomas of other organs was also stained. Sarcomas, lymphomas, and other tumors of nonepithelial origin were constantly negative. Staining of some normal epithelial cells was also observed. Among the fetal tissue tested, the antibody reacted with pancreatic ducts and the small intestine. The antibody recognized metastatic carcinoma cells in peritoneal effusions. On the basis of its tissue distribution, the antigenic determinant defined by the AR-3 monoclonal antibody was called CAR-3. The monoclonal AR-3 did not cross-react with partially purified preparations of carcinoembryonic antigen, gastrointestinal carcinoma antigen, or the human milk fat globule antigen. The AR-3 MAb appear, thus, to broaden the number of available reagents for histopathological diagnosis of carcinomas.

Estrogen- and tamoxifen-induced rearrangement of cytoskeletal and adhesion structures in breast cancer MCF-7 cells.

The cytoskeleton, the shape, and the adhesion complexes of MCF-7 breast carcinoma cells have been studied by fluorescence, phase contrast, and interference reflection microscopy. Cells have been grown in media containing different concentrations of estrogen and with or without the addition of the antiestrogen tamoxifen. The pattern of actin microfilaments and keratin intermediate filaments (tonofilaments) and the distribution of adhesion areas change as a function of the estrogen concentration. When cells are cultured in estrogen-deprived medium, they appear roundish and flattened and adhere firmly to the substratum, with multiple vinculin-positive adhesion plaques at their ventral surface. Upon stimulation with estrogen, these cells display pseudopodial cytoplasmic protrusions and ruffling membranes; in interference reflection microscopy the adhesion areas are mostly localized in these projections. A rearrangement of microfilaments and of tonofilaments in the cell projections and the formation of a dense network of keratin fibers takes place. Tamoxifen affects cellular shape and cytoskeletal arrangement in a way similar to that induced by estrogen. An effect of estrogen-receptor stimulation on the adhesion structures and on the rearrangement of intermediate and actin filaments (and accordingly of the shape and internal structure of breast cancer cells) can be suggested. Such an effect might be direct or mediated through unknown mechanisms; it seems, however, to be independent of the well known estrogenic effect on cell proliferation.

Tumor types derived from epithelial and myoepithelial cell lines of R3230AC rat mammary carcinoma.

Epithelial and myoepithelial cells coexist in the rat R3230AC mammary tumor. To test the hypothesis that these two cell types constitute interactive but independent neoplastic populations, we obtained in vitro cell lines with epithelial or myoepithelial patterns and transplanted them in syngeneic animals. One stabilized line (EPI) and four cloned lines (A, C, D, E) with epithelial characteristics, confirmed by positive reactions for keratins in immunocytochemical and immunoblot tests, constantly gave rise in vivo to carcinomas, which, however, lacked structural and functional patterns typical of the original tumor. A fusiform shape and immunocytochemical characteristics of myoepithelial cells were observed in three clones (H, I, L), which in vivo gave rise to sarcomatous and mixed carcinosarcomatous neoplasms. These data are consistent with the above hypothesis and indicate that breast carcinomas derive from epithelial cells, while sarcomatous and carcinosarcomatous neoplasms can originate from myoepithelial cell proliferation. This study provides data suggesting myoepithelial cell involvement in the development of pathological entities occurring in the human breast and displaying mixed epithelial and stromal neoplastic components, i.e., cystosarcoma phylloides and sarcomatous metaplasia in carcinomas.

111In-labeled 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid-lys(8)-vasotocin: a new powerful radioligand for oxytocin receptor-expressing tumors.

We developed a radioactive ligand for tumors expressing oxytocin receptors (OTRs) by linking the chelating agent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) to Lys(8)-vasotocin (LVT), an analogue of oxytocin with high affinity for OTRs. The new reagent (DOTA-LVT) retained high affinity for human OTRs, as proved by in vitro affinity binding to cells endogenously expressing OTRs, such as MCF7 breast carcinoma and MOG-U-V-W glioblastoma cells lines, as well as to transiently transfected COS7 cells. In in vivo experiments, DOTA-LVT carrying (111)In showed specific binding activity to OTR-positive TS/A mouse mammary tumors. The present study opens new perspectives for imaging and, possibly, therapy of OTR-positive human tumors such as breast and endometrial carcinomas, neuroblastomas, and glioblastomas.

A plasmin-derived hexapeptide from the carboxyl end of osteocalcin counteracts oxytocin-mediated growth inhibition [corrected] of osteosarcoma cells.

We have previously described the presence of the functional plasminogen activator system on the surfaces of bone neoplastic cells and the fact that plasmin specifically cleaves bone matrix protein osteocalcin (OC). The cleavage of OC to NH2-midterminal (1-44) and COOH-terminal RFYGPV hexapeptide (44-49) proceeds with detachment of both products from bone mineral. Because the sequence of OC-derived hexapeptide (HP) is nearly identical to the E2 region of the oxytocin receptor (OTR), we set out to ascertain whether the HP interferes with the osteosarcoma (OS)-associated oxytocin (OT) system. We documented the presence and functional activity of OTRs in several OS cells by means of (a) OT-mediated inhibition of OS growth; (b) expression of OTR mRNA by means of reverse transcription-PCR; (c) immunofluorescence staining with IF3 monoclonal antibody specific for human OTR; and (d) saturation binding and Scatchard analysis of OT binding to the receptors of isolated membranes or intact OS cells. Although we could not demonstrate direct binding of HP to OT, the presence of HP in cultures of OS cells antagonizes the inhibitory effect of OT on these cells. Additionally, in competitive binding assays, the HP effectively competes with binding of OT to its cognate receptors. The results indicate the existence of an OTR/OT system in tumor cells of bone origin. Suggested plasminogen activator-OC-OTR/OT interactions may have an effect on the regulation of cell proliferation within the bone tissue as well as properties of the extracellular matrix surrounding the tumor foci in the bone.

Quantitation of somatostatin receptor type 2 gene expression in neuroblastoma cell lines and primary tumors using competitive reverse transcription-polymerase chain reaction.

We previously reported the presence of somatostatin (SS-14)-binding sites in a wide panel of human neuroblastoma (NB) tumor cell lines. Given that the adrenal gland and its relative embryonal and adult tumors express an abundance of mRNA for somatostatin receptor type 2 (sst2) mRNA, we studied the quantitative expression of sst2 in 6 NB cell lines and 15 primary tumors using competitive reverse transcription (RT)-PCR. This method uses an insertion mutant of the target gene as a competitor for the RT-PCR reaction, thus allowing exact quantitation of sst2 mRNA abundance. We found expression of specific transcripts for sst2 in all of the NB cell lines and tumors investigated (range, 9 x 10(5)-4 x 10(9) molecules/microg RNA). In NB cells, the expression of sst2 was highly correlated with SS-14-binding sites (R = 0.93). In primary tumors, sst2 was positively related to the expression of the neuroendocrine marker secretogranin II (P < 0.05) and negatively related to N-myc amplification (a poor prognostic factor, P < 0.005) and metastatic dissemination (P < 0.05). In addition, Kaplan-Meier curves indicate that sst2 expression is positively related to survival (P = 0.01). In a patient with stage IVs disease (a spontaneously regressing form), we found the highest sst2 expression (4 x 10(9) molecules/microgram RNA), a value relatively similar to that of normal adrenal. In conclusion, these data indicate that quantitation of sst2, as assessed with competitive RT-PCR, could represent a new prognostic tool in the neuroendocrine tumor NB. Since sst2 recognizes octreotide with high affinity, these findings could also have both diagnostic and therapeutic value.

Oxytocin enhances myoepithelial cell differentiation and proliferation in the mouse mammary gland.

Using immunocytochemistry and electron microscopy, we demonstrate that oxytocin (OT) exerts a trophic effect on its target myoepithelial cells in the mammary gland. In vitro, in organotypic cultures of mouse mammary gland, we examined proliferation and differentiation of the different cell types induced by OT added to the medium. In vivo, we studied the effect of OT on the structure and cell composition of developing glands. Uptake of 5-bromo-2'-deoxyuridine was used as proliferation marker, while antibodies to smooth muscle alpha-actin (specific for myoepithelial cells) and keratin (MoAb AE1; selective for epithelial cells) were used to identify differentiated cell types. By electron microscopy, we studied structural modifications induced by OT on the extreme projections of the developing gland (sc end buds). The results indicate that OT induces myoepithelial cell differentiation and proliferation, enhancing the effect of mammotrophic hormones in nonlactating mouse mammary gland. A less marked effect was observed in luminal epithelial cells. No significant effect of OT alone was detected in cultured glands from unprimed animals.