Malignancy-associated changes in breast tissue detected by image cytometry.

In several tissues, nuclear differences have been described in normal-appearing cells from patients with invasive carcinomas compared to cases without invasive carcinoma, a phenomenon known as malignancy-associated changes (MACs). The aim of this study was to determine the presence of malignancy-associated changes in breast tissue. Image cytometry was performed on Feulgen stained tissue sections of patients with usual ductal hyperplasia with (n = 30) or without (n = 41) adjacent invasive breast carcinoma. Nuclear features of normal-appearing cells as well as of usual ductal hyperplastic cells were separately compared between the two groups. Many features of normal-appearing epithelial cells were significantly different between cases with and without invasive cancer. Significant differences were also found by measuring ductal hyperplastic nuclei instead of normal-appearing nuclei. Cases with or without cancer could be distinguished with a classification accuracy of 80% by discriminant analysis using 2 nuclear features derived from ductal hyperplastic cells. In conclusion, image cytometry on breast tissue sections shows that malignancy-associated changes can be found in normal as well as in usual ductal hyperplastic breast cells. This could be clinically relevant for the detection of occult breast cancer, for the prediction of risk in these lesions, and to monitor the effect of chemopreventive agents.     

Long-term organ culture of mouse mammary gland

Summary A method for maintaining mouse mammary gland in organ culture for periods of at least 30 days is described. Strips of the number four mammary glands were cultured in individual tubes while fully submerged in Medium 199 supplemented with insulin, aldosterone, ovine prolactin and bovine growth hormone. Exchange processes were aided by slowly rotating the tubes during culture. Mammary tissue from midpregnant BALB/c and virgin GR/A mice was induced to undergo lobulo-alveolar development, secrete and remain differentiated and metabolically active for the period of culture. Cells of both the ductal and alveolar epithelium continued to synthesize DNA and divide. The submerged roller-tube culture allows the use of larger pieces of tissue than can be accommodated in static culture, and the technique may prove applicable to the culture of a variety of tissues.     

3-Dimensional Resin Casting and Imaging of Mouse Portal Vein or Intrahepatic Bile Duct System

In organs, the correct architecture of vascular and ductal structures is indispensable for proper physiological function, and the formation and maintenance of these structures is a highly regulated process. The analysis of these complex, 3-dimensional structures has greatly depended on either 2-dimensional examination in section or on dye injection studies. These techniques, however, are not able to provide a complete and quantifiable representation of the ductal or vascular structures they are intended to elucidate. Alternatively, the nature of 3-dimensional plastic resin casts generates a permanent snapshot of the system and is a novel and widely useful technique for visualizing and quantifying 3-dimensional structures and networks.A crucial advantage of the resin casting system is the ability to determine the intact and connected, or communicating, structure of a blood vessel or duct. The structure of vascular and ductal networks are crucial for organ function, and this technique has the potential to aid study of vascular and ductal networks in several ways. Resin casting may be used to analyze normal morphology and functional architecture of a luminal structure, identify developmental morphogenetic changes, and uncover morphological differences in tissue architecture between normal and disease states. Previous work has utilized resin casting to study, for example, architectural and functional defects within the mouse intrahepatic bile duct system that were not reflected in 2-dimensional analysis of the structure^1,2, alterations in brain vasculature of a Alzheimer''s disease mouse model³, portal vein abnormalities in portal hypertensive and cirrhotic mice⁴, developmental steps in rat lymphatic maturation between immature and adult lungs⁵, immediate microvascular changes in the rat liver, pancreas, and kidney in response in to chemical injury⁶.Here we present a method of generating a 3-dimensional resin cast of a mouse vascular or ductal network, focusing specifically on the portal vein and intrahepatic bile duct. These casts can be visualized by clearing or macerating the tissue and can then be analyzed. This technique can be applied to virtually any vascular or ductal system and would be directly applicable to any study inquiring into the development, function, maintenance, or injury of a 3-dimensional ductal or vascular structure.     

Epithelial-myoepithelial carcinoma of the parotid. A case of ductal-predominant presentation with cytologic, histologic and ultrastructural correlations

BACKGROUND: The cytologic features of the usual type of epithelial-myoepithelial carcinoma (EMC) of the parotid, with myoepithelial cell predominance, is well described in the cytology literature. In contrast, the cytologic features of ductal-predominant-type EMC has not yet been reported. CASE: An 82-year-old male presented with a 2.7-cm parotid mass of two years' duration. Fine needle aspiration smears stained with Diff-Quik showed cohesive tissue fragments outlined by metachromatic fibrils scattered in abundant, smooth, bluish background material. Ultrafast Papanicolaou stain revealed sharply outlined, large ductal cells with smooth, round to oval nuclei, prominent nucleoli and abundant vacuolated cytoplasm; the cells were arranged tridimensionally in occasional follicles that contained thick secretions. Neoplastic myoepithelial cells were occasionally seen at the periphery of tissue fragments, most commonly hidden underneath the neoplastic ductal epithelium at a slightly different focal plane; the cells had small, oval, dark nuclei and inconspicuous cell borders. The nuclear area and cell size of the neoplastic ductal cells was two and three times, respectively, that of neoplastic myoepithelial cells. CONCLUSION: EMC, depending on the ratio of ductal to myoepithelial cell components, has different cytologic presentations. This case illustrates the ductal-predominant presentation of EMC.     

Expression of the Na(+)/I(-) symporter in invasive ductal breast cancer

The function of the sodium iodide symporter (Na(+)/I(-), (NIS), a membrane protein that mediates iodide transport into cells, is the best described in the thyroid cells. NIS is also found in mammary cells during lactation and in breast carcinoma cells. The aim of this study was evaluation of incidence and grade of NIS expression in invasive ductal breast cancer. Immunohistochemistry using a panel of antibodies against NIS was carried out in surgical paraffin-embedded tissue obtained from 50 patients with invasive ductal breast carcinoma. NIS expression was found in 45 (90%) cases. The demonstration of NIS expression in breast carcinoma cells may provide a novel approach to its diagnosis and treatment.     

Sacrificial Bioprinting of a Mammary Ductal Carcinoma Model

Cancer tissue engineering has remained challenging due to the limitations of the conventional biofabrication techniques to model the complex tumor microenvironment. Here, the utilization of a sacrificial bioprinting strategy is reported to generate the biomimetic mammary duct‐like structure within a hydrogel matrix, which is further populated with breast cancer cells, to model the genesis of ductal carcinoma and its subsequent outward invasion. This bioprinted mammary ductal carcinoma model provides a proof‐of‐concept demonstration of the value of using the sacrificial bioprinting technique for engineering biologically relevant cancer models, which may be possibly extended to other cancer types where duct‐like structures are involved.     

Gynecomastia: suction lipectomy as a contemporary solution

Suction lipectomy is adapted for the correction of gynecomastia. Previous attempts using suction lipectomy for gynecomastia still required the use of sharp dissection for removal of the glandular breast tissue as well as excision of redundant skin. With this new technique, gynecomastia is corrected solely with the use of suction lipectomy. The technique is successful if the gynecomastia is due to excess fat or parenchymal hypertrophy. A 7-mm cannula is inserted first, to remove the adipose tissue. Then a 2.4-mm cannula is used to remove the glandular and ductal tissue. The 7-mm cannula is then reinserted to remove subareolar parenchyma and to make final contour adjustments. The surrounding subcutaneous tissue is easily undermined to avoid a saucer deformity and to allow for skin contraction. Patients return to full activities in 48 hours. A compressive garment is worn for 4 to 6 weeks. The results of 10 patients are discussed.     

Stain–Decolorize–Stain (SDS): a new technique for multiple staining

Multiple staining of more than one gene/antigen on a single tissue section is an indispensable tool in cell and tissue research. However, most of the available multiple staining techniques have limitations, and there has been no technique to simultaneously visualize and distinguish tissue antigens, nucleotide sequences and other chemical compounds on the same slide. Here, we present a practical and economic multiple stain technique, with which multiple cellular components including mRNA (with in situ hybridization), antigen epitope (with immunohistochemistry) and chemical molecules (with histochemistry) can be stained on a single tissue section to study their relationship. In addition, this technique also offers the possibility to evaluate morphology with an H&E staining on the same sections. We used the placenta, pancreas, breast ductal carcinoma, colon adenocarcinoma, cerebellum, tonsil and heart tissue sections to evaluate the applicability of this new technique. The sensitivity and specificity of the technique have been tested, and an optimal protocol is recommended. Its applications in surgical pathology and research are discussed. This technique offers a novel tool to evaluate the relationship among multiple components at the same or adjacent locations to meet the needs of pathology diagnosis and research.     

Differential expression of laminin chains in the human major salivary gland

Laminin represents a macromolecule family. The heterotrimeric isoforms of laminin can be determined by immunohistochemical demonstration of the single chains. The laminin chain heterogeneity of the basement membrane in adult human major salivary glands was evaluated in relation to cellular differentiation of the epithelia and the stromal compartment. Monoclonal antibodies to the laminin alpha1, alpha3 (BM165) chains and epiligrin reacted with the basement membranes of serous and mucous acini and of intercalated, striated and excretory ducts. As evidenced by a double-labelling technique, the alpha2 chain showed a spatial association with the myoepithelium of the acini, whereas the ductal basement membranes containing no myoepithelial cells were negative. Almost exclusively, beta1 chain was detected in acinar basement membrane, beta2 chain whereas in ductal basement membrane. gamma2 chain is a unique chain belonging to the laminin-5 isoform. It was restricted to the ductal basement membrane. alpha1, alpha2, beta1, beta2 and gamma1 chains were detected in nerves of salivary tissue and alpha1, alpha3, beta1, beta2 and gamma1 chains and epiligrin in blood vessels. Our results indicate that the acinar ductal unit contains basement membranes with different isoforms, which relate to cell differentiation and cell function. © 1998 Chapman & Hall     

Rapid purification of human ductal cells from human pancreatic fractions with surface antibody CA19-9

Generating human insulin-secreting cells for cell therapy of diabetes represents a highly competitive world challenge. Human ductal cells can give rise to islets in vivo and in vitro. The goal of this study was to devise a rapid sorting method to highly purify human ductal cells from pancreatic tissue using a pan-ductal membrane antibody carbohydrate antigen 19-9 (CA19-9). Human pancreatic sections confirmed antibody specificity. The human exocrine fraction (30% ductal cells) was sorted with magnetic bead technology or by FACS. Immunocytochemistry post-sorting determined ductal cell content. The manual magnetic bead technique resulted in 74%+/-2 (n = 4) CA19 positive cells. Whereas the automated AutoMACS technique (n = 5) yielded 92.6%+/-0.5 CA19-9 positive cells with only a minor beta cell contamination (0.2%+/-0.03); cell yield post-sorting was 12.9%+/-2.5 (1.69+/-0.41 x 10(6) cells) with 51.7%+6.5 (n = 5) viability post-sorting. The FACS (n = 6) resulted in 97.1%+/-0.82 CA19-9 positive cells, a cell yield of 25.5%+/-5.6 (5.03+/-1.0 x 10(6)), with 72.1%+/-6.1 viability post-sorting.     

Use of specimen mammography-guided FNA (fine-needle aspirates) for flow cytometric multiple marker analysis and immunophenotyping in breast cancer

A pilot study of a novel translational research method to simultaneously assay multiple molecular markers and DNA in fine-needle aspirates (FNA) of mammographically detected breast lesions is described. Specimen mammography-guided 20-gauge FNAs obtained from 86 lesions and 22 areas of normal tissue were analyzed by multiparameter flow cytometry for DNA content, her2/neu, transforming growth factor alpha (TGF alpha), and the epithelial marker cytokeratin (CK) simultaneously. Epithelial cell her2/neu positivity was detected in 12 of 44 (27%) of invasive ductal carcinomas and 3 of 9 (33%) ductal carcinoma in situ (DCIS), 10 of 30 (33%) benign lesions, and 4 of 22 (18%) normal tissue aspirates. All lesions and normal tissue showed a similar positive rate for TGFalpha ranging from 61 to 76%. The CK(+)TGF alpha(-)her2/neu(+) immunophenotype was more frequently positive in aneuploid tumors (22%) than all other lesions (7%) (P < 0.05). Specimen mammography-guided FNAs provide fresh cells for flow cytometric multiple marker analysis and immunophenotyping of clinically occult breast lesions and normal tissue.     

Progesterone involvement in breast development and tumorigenesis--as revealed by progesterone receptor "knockout" and "knockin" mouse models

In light of recent clinical trials, the debate concerning the risks and benefits of progestin-based postmenopausal hormone replacement therapy (HRT) has reached a renewed level of urgency. Irrespective of the position taken, the consensus is that more basic research needs to be performed to address progesterone's fundamental role in mammary development and tumorigenesis. Towards this end, the progesterone receptor knockout (PRKO) mouse demonstrated that progesterone is essential for pregnancy-associated mammary gland ductal side-branching and alveologenesis and that these morphological changes are dependent on progesterone-induced mammary epithelial proliferation. Importantly, the PRKO mouse showed that the progesterone-proliferative signal significantly contributes to mammary tumor susceptibility in an established mammary tumor model. Insight into the cellular mechanism(s) by which progesterone affects mammary morphogenesis has been disclosed by a new PR-LacZ knockin mouse, which revealed that PR's spatial expression pattern undergoes precise choreographed distributional changes that precede key stages in postnatal mammary development. In the case of early pregnancy, the segregation of cells undergoing progesterone-induced proliferation from those that express PR implicates a paracrine mode of action for progesterone-induced mammary epithelial proliferation, whereas the preparturient decline of PR expression underscores the need to remove this signal for full functional differentiation of this tissue. Our findings support the proposal that the mammary gland's normal response to the progesterone-signal is dependent upon specific spatial organizational patterns of PR expression and that derailment in these cellular processes may contribute to abnormal mammary development, including cancer. This review concludes by emphasizing the need to identify the downstream molecular targets that mediate progesterone's effects in this tissue. Identification of such targets will not only enhance our mechanistic understanding of progesterone's role in mammary development and cancer, but may also facilitate the formulation of new design strategies in breast cancer diagnosis and/or treatment.     

Histone H3 mRNA in situ hybridization for identifying proliferating cells in human pancreas, with special reference to the ductal system

In general, the incidence of proliferating cells parallels that of carcinogenesis. We have investigated proliferating activity and phenotype expression in epithelial cells in normal tissue, mucinous metaplasia and ductal adenocarcinoma of the pancreas. Twenty-eight resected pancreases (15 cases of pancreatic ductal adenocarcinoma and 13 cases of other diseases) were examined. Formalin-fixed, paraffin-embedded tissue sections were examined for proliferating cell activity using histone H3 mRNA in situ hybridization and immunostaining for Ki-67. In the normal pancreas, the labelling indices for proliferating cells were low and no generating zone was found. The following progressive increase was found in the labelling indices: normal ductal epithelium < mucinous metaplasia without papillary hyperplasia < mucinous metaplasia with papillary hyperplasia < ductal carcinoma. In the pancreatic ductal adenocarcinomas, the S-phase fraction, as defined by the ratio H3-mRNA-labelling index/Ki-67-labelling index, increased as the degree of differentiation decreased. Mucinous metaplasia with papillary hyperplasia showed organoid differentiation toward pyloric mucosa. If used in combination with other proliferative markers on paraffin-embedded tissue sections, histone H3 mRNA in situ hybridization could open broader perspectives on the biology of cell proliferation in the pancreatic ductal system.     

Immunohistochemical demonstration of the carbonic anhydrase isoenzymes I and II in pancreatic tumours

Summary The location of carbonic anhydrase (CA) isoenzymes I, II and VI in normal and neoplastic pancreatic tissue was studied using polyclonal antisera and the immunoperoxidase technique. Samples were obtained from patients with well-differentiated (n = 4), moderately differentiated (n = 1) and poorly differentiated (n = 4) ductal adenocarcinomas, cystadenocarcinoma (n = 2), adenosquamous carcinoma (n = 1), acinar adenocarcinoma (n = 1), gastrinoma (n = 3), insulinoma (n = 3) and glucagonoma (n = 1). The control specimens were from a patient with traumatic laceration of the pancreas. The normal and malignant endocrine tissue showed intense positive staining for CA I localized in the cells expressing glucagon. In the exocrine pancreatic tissue, CA II was detected in the normal and neoplastic ductal epithelium. No specific staining was detected with anti-CA VI serum in either normal or malignant tissue.     

Sclerosing polycystic adenosis of the left parotid gland: report of a case with fine needle aspiration cytology

BACKGROUND: Sclerosing polycystic adenosis (SPCA) of major salivary glands is a rare recently described entity. We report a case of SPCA of the left parotid gland, including the cytologic and histopathologic findings. CASE: A 20-year-old man presented with a left parotid mass that had been growing slowly for 3 years. Fine needle aspiration cytology showed many syncytial cell clusters of variable size and some ductal structures with an inflammatory background. The cells forming syncytial clusters were large and polygonal, with abundant, eosinophilic, granular or lacelike cytoplasm. Apocrine differentiation with decapitation secretion was commonly seen. The ductal cells had a relatively high nuclear/cytoplasmic ratio, with granular cytoplasm. Grossly, the 5-cm lesion was a discrete, pale, cystic nodule embedded within the parotid gland parenchyma. Microscopically, the lesion was a nonencapsulated, circumscribed mass of sclerotic and hyalinized, collagenous tissue with lymphoplasmacytic infiltration. Sclerosing adenosis and cystic ducts with frequent apocrinelike cells were commonly seen. Some acinar cells contained eosinophilic, intracytoplasmic granules of various sizes. CONCLUSION: The presence of syncytial clusters with apocrine metaplasia and ductal structures in a lymphoplasmacytic background should suggest a diagnosis of SPCA of a major salivary gland.     

Ultrasonographic and scintigraphic studies of "hot" autonomic thyroid nodules

Scintigraphic and gammagraphic (gamma-camera) techniques have been applied in diagnosing "hot" non-TSH dependent thyroid nodules. On the basis of frequent uneven accumulation of isotopic tracer observed in scintigraphic studies it was concluded that the structure of these nodules is heterogeneous. In some cases paradoxical patterns have been obtained following the dynamic tests of suppression and stimulation of the thyroid: after the administration of thyroid preparations paranodular thyroid tissue appears, while TSH administration causes an increase in the iodine uptake by the nodule. Ultrasonographic and gamma-camera studies have been carried out in 50 cases of "hot" autonomic thyroid nodules previously diagnosed by scintigraphy. Ultrasonographic studies revealed a weaker echogenicity in some cases and an intensified echo in the other. Sometimes by using this technique it was possible to demonstrate the occurrence of heterogeneous structures suggesting a cystic degeneration of the nodule. Paranodular thyroid tissue, invisible in scintigraphic scanning, could usually be observed by applying gamma-camera. It was concluded that both gamma camera and ultrasonographic studies confirm heterogeneous structure of intranodular tissue producing paradoxical patterns in scintigraphic scanning, with the latter technique usually aiding the detection of paranodular thyroid tissue without the necessity of application of the TSH test.