Nuclear localization of MUC1 extracellular domain in breast, head and neck, and colon cancer.

BACKGROUND: The glycoprotein MUC1 is overexpressed and underglycosylated in cancer cells. MUC1 is translated as a single polypeptide that undergoes autocleavage into 2 subunits (the extracellular domain and the cytoplasmic tail), and forms a stable heterodimer at the apical membrane of normal epithelial cells. The MUC1 cytoplasmic tail localizes to the cytoplasm of transformed cells and is targeted to the nucleus. AIMS: To study the expression of the MUC1 extracellular subunit in cell nuclei of neoplastic breast, head and neck, and colon samples. MATERIALS AND METHODS: 330 primary tumor samples were analyzed: 166 invasive breast carcinomas, 127 head and neck tumors, and 47 colon tumors; 10 benign breast disease (BBD) and 40 normal specimens were also included. A standard immunohistochemical method with antigen retrieval was performed. Nuclear fractions from tissue homogenates and breast cancer cell lines (ZR-75, MDA-MB-231, MCF7, and T47D) were obtained and analyzed by Western blotting (WB). The anti-MUC1 extracellular subunit monoclonal antibody HMFG1 was used for immunohistochemistry. RESULTS: 37/166 breast cancer specimens, 5/127 head and neck cancer specimens, 2/47 colon cancer samples, and 3/10 BBD samples showed immunohistochemical staining at the nuclear level. No nuclear reaction was detected in normal samples. By WB, breast and colon cancer purified nuclear fractions showed reactivity at 200 kDa in 3/30 breast and 3/20 colon cancer samples as well as purified nuclear fractions obtained from breast cancer cell lines. CONCLUSIONS: This study shows that the MUC1 extracellular domain might be translocated to the cell nucleus in breast, head and neck, and colon cancer as well as BBD.

Differentiating the two main histologic categories of fibroadenoma tissue from normal breast tissue by using multiphoton microscopy.

Multiphoton microscopy has become a novel biological imaging technique that allows cellular and subcellular microstructure imaging based on two-photon excited fluorescence and second harmonic generation. In this work, we used multiphoton microscopy to obtain the high-contrast images of human normal breast tissue and two main histologic types of fibroadenoma (intracanalicular, pericanalicular). Moreover, quantitative image analysis was performed to characterize the changes of collagen morphology (collagen content, collagen orientation). The results show that multiphoton microscopy combined with quantitative method has the ability to identify the characteristics of fibroadenoma including changes of the duct architecture and collagen morphology in stroma. With the advancement of multiphoton microscopy, we believe that the technique has great potential to be a real-time histopathological diagnostic tool for intraoperative detection of fibroadenoma in the future.

Technology of building an expert system based on a set of quantitative features of tumor cell nuclei for diagnosing breast cancer.

The technology of building an expert system for diagnosing malignant nature of invasive tumors of the mammary gland based on a set of quantitative features of the cell nuclei has been developed. Its peculiarity was the presence of weighting coefficients in all the features. Quantitative features were obtained by transforming the initial morphometric data with the help of simple (evaluation of mean values and building of histograms) and complex (regression analysis) mathematical operations. The expert system consisted of one-dimensional X-matrix used for investigations and two-dimensional standard S-matrix. The X-matrix elements were assigned for filling with the quantitative features of the studied sample with a nonestablished diagnosis. The S-matrix elements contained threshold values of quantitative features from the system of diagnostic decision criteria for malignant forms of diseases and their weighting coefficients. Threshold values of nuclear features (larger or smaller) were determined taking into account the range of their values in the groups of malignant and benign pathology. Significance of quantitative features in diagnosing diseases has been assessed. The presence of weighting coefficients allowed diagnosing malignant and benign pathology in a quantitative form by the diagnostic index value. Diagnostic index was calculated by the sum of weighting coefficients of features of the studied sample, which fell within the range of system of the S-matrix diagnostic decision criteria. Clinical trials revealed high efficiency of the developed approach while diagnosis of breast cancer invasive forms at a preoperative stage.

[Heterogeneity of angioarchitecture and their hemodynamic changes in benign and malignant breast tumors].

OBJECTIVE: To explore the differences between the angioarchitecture, hemodynamics, ultrastructure of neovasculr endothelial cells, and vascular distribution in different perfusion regions in benign and malignant breast tumors. METHODS: 30 cases of breast carcinoma (33 lesions) and 30 cases of breast fibroadenoma (34 lesions) were examined by contrast enhanced microvascular imaging (MVI), and perfusion indexes were collected both inside and at the margin of each focus according to time-intensity quantitative analysis, including peak intensity (PI), area under the curve (AUC), time to peak (TTP) and wash-out time (WOT). The ultrastructure of neovascular endothelial cells was examined by transmission electron microscopy. The expression of CD34, VEGF, Flk-1/KDR in both two groups were detected by immuhistochemistry. RESULTS: Significant differences were found between the two groups characterized with filling defect, vascular distortion, dilatation and uneven enhancement. Most of the curves of malignant group (87.9%, 29/33) ascended rapidly and dropped slowly while those of the benign group (79.4%, 27/34) ascended slowly and dropped rapidly. The AUC and WOT of malignant tumor group were significantly higher than those of benign group, while the PI and TTP had statistically no significant difference. In the malignant tumor group, PI, AUC and WOT collected from the margin of foci were significantly different from those collected inside the foci, however, there was no significant difference in the benign group. The margin of foci was characterized with dilated and distorted vessels, and the center of the foci was occupied by narrow or occluded blood vessels, sometimes with contracted endothelial cells and pericytes. Abundant microvascular areas located at the margin of foci. The ultrastructure of endothelial cells in the newly formed blood vessels of malignant group showed strong ability to divide, which was different from normal endothelium cells. CONCLUSION: The perfusion pattern, mode of time-intensity curve, mean perfusion parameter and variation of regional perfusion parameters provide a valuable diagnostic basis in distinguishing benign and malignant breast tumors. The density, morphology, distribution, structure and function of newly formed microvessels in tumor foci are also crucial factors when tumors are assessed by imaging examination.

Morphologic changes in fibroadenoma of breast due to chickenpox: a case report with suspicious cytology in fine needle aspiration smears.

BACKGROUND: Fibroadenomas with stromal giant cell reaction have been described in the literature, but cytologic atypia including giant cell reaction due to chickenpox giving rise to suspicious cytology has not been reported. CASE REPORT: A 25-year-old woman, recovering from chickenpox, presented with a 1.5 x 1.5-cm mass in the lower outer quadrant of her right breast. Fine needle aspiration smears showed sheets of benign ductal cells with overlapping myoepithelial cells and many bipolar bare nuclei. Cells showing nuclear enlargement, prominent nucleoli and multilobated or multinucleated giant cell formation occurred in separate sheets or dispersed among groups of benign ductal cells. Cytodiagnosis was suspicion for malignancy; excision biopsy was advised. Histopathologic examination showed fibroadenoma with evidence of epithelial hyperplasia, nuclear enlargement and multilobated giant cell formation. Atypical ductal cells, including the giant cells, were immunohistochemically positive for epithelial membrane antigen, estrogen receptor and progesterone receptor and negative for smooth muscle actin, indicating epithelial origin. Both cytologic and histologic specimens showed focal positive reaction with HSV-1 and HSV-2 antibodies. Ultrastructural examination of aspirated material revealed cytoplasmic viral particles with characteristic surface projections. CONCLUSION: Herpes zoster virus can produce morphologic alteration mimicking a malignancy. Pathologists should be aware of these changes to avoid a false positive diagnosis.

Ultrastructural immunostaining of infiltrating ductal breast carcinomas with the monoclonal antibody H: a comparative study with cytokeratin 8.

The monoclonal antibody H (mAbH) detects an epitope consisting of an O-linked N-acetyl glucosamine (O-GlcNAc) and neighboring amino acids. This epitope has been found by using extracts from the MCF-7 human breast carcinoma cell line in immunoblotting experiments, on cytokeratin 8 (CK8) and 5 other polypeptides. In the present study, a double immunogold method was applied for the colocalization of CK8 and mAbH epitope on epoxy thin sections in 18 cases of infiltrating ductal breast carcinomas (IDBC) and in 6 cases of fibroadenomas, to study the accurate subcellular distribution of CK8 in breast cancer cells, as compared to the 5 polypeptides, recognized by mAbH. Furthermore, a detailed quantitative evaluation of the double immunolocalization over the cellular compartments of cancer cells was undertaken with the aid of a computerized image analysis system and the results were assessed statistically. The distribution pattern of CK8 and the mAbH epitope in the neoplastic mammary epithelial cells was similar in IDBC as compared to fibroadenomas, while the gold labeling intensity of these epitopes differed over the cellular compartments between malignant and benign biopsies. The results reveal the significance of the role of CK8 and O-GlcNAc glycosylation in the biology of the neoplastic mammary cells in vivo, determining their malignant potential.

Differentiation of benign and malignant breast tumors using Doppler spectral parameters including acceleration time index.

Differentiation of breast cancer has been receiving increasing attention in attempts to minimize the numbers of excisional biopsies for benign tumors. Doppler spectral parameters, such as peak systolic velocity (PSV) and pulsatility index (PI), have been used but these are unable to differentiate breast cancers and benign tumors with adequate levels of specificity and sensitivity. The aim of this study was to determine whether the acceleration time index (ATI) could reliably differentiate breast tumors. In 102 breast masses, Doppler ultrasonography (US) was performed with a LOGIQ700MR. PSV, PI and ATI were calculated from intratumoral flow. Forty-eight malignant tumors were identified by histologic evaluation, 13 benign tumors were diagnosed by fine needle aspiration biopsy (FNAB) and 41 benign masses were diagnosed by characteristic gray-scale findings and long-term (>18 months) follow-up examinations. Doppler signals were detected in 35 of 54 (64.8%) benign lesions and in 46 of 48 (95.8%) breast cancers. PSV and PI value showed no statistical significance between malignant and benign lesions (PSV; p=0.1147, PI; 0.1506). ATI values were lower in benign lesions (p<0.0001). An ATI value of 0.14 seems to be the optimum threshold for the differentiation of benign and malignant lesions (79.4% sensitivity, 87.2% specificity). No malignant lesions were seen when the ATI value was <0.14 and the PI value was <1.1. ATI was found to be the most useful parameter for differentiating benign breast tumors from malignant ones.

Fine needle aspiration biopsy of the breast. Value of nuclear morphometry after different sampling methods.

OBJECTIVE: To study the potential of nuclear morphometry in supporting the interpretation of fine needle aspiration biopsy (FNAB) samples of the breast fixed in 50% ethanol and centrifuged on slides. STUDY DESIGN: Computerized morphometry was used to outline the nuclei of breast epithelial cells in breast cancer, fibroadenoma and fibrocystic disease. The diagnoses were histologically confirmed. We applied 2 different sampling methods (measurements done on cell groups and on free cells). RESULTS: The mean nuclear area of cell groups of malignant samples (23) varied from 42 to 125 microns 2, in fibroadenomas from 30 to 50 microns 2 and in fibrocystic disease from 26 to 57 microns 2. The mean nuclear area of free cells varied as follows: cancer, 66-181 microns 2; fibroadenoma, 33-70 microns 2; fibrocystic disease, 35-60 microns 2. Apocrine metaplasia was excluded from comparison on a morphologic basis. CONCLUSION: The study suggests that if the mean nuclear area of cell groups is < 42 microns 2, the lesion is probably benign; if > 57 microns 2, and apocrine metaplasia is excluded, malignancy should be considered. The differential diagnosis between carcinoma and fibroadenoma could be based on free cells: mean area of free cell nuclei < or = 65 microns 2 suggested a benign lesion, and of > or = 71 microns 2 suggested a malignant lesion. Morphometric nuclear size features (exemplified by nuclear area) appeared efficient in distinguishing between malignant and benign lesions when measured from free cells and cell groups.

Pseudoangiomatous hyperplasia of mammary stroma: a case of pure type after removal of fibroadenoma.

A case of pure pseudoangiomatous hyperplasia of the mammary stroma after removal of a fibroadenoma is described. The lesion, which was found in the right breast of a 40-year-old woman, was a well-circumscribed non-encapsulated, rubbery, lobulated mass measuring 40x40x35 mm. Histologically, it consisted predominantly of a proliferation of spindle cells with interanastomosing vascular-like arrangements in the interlobular or interductal stroma. Neither cytological atypia nor mitotic figures were observed. The stroma contained abundant collagen with focal hyalinization. Focally, epithelial elements showed mild ductal hyperplasia. There was no fibroadenoma. Immunohistochemically, the spindle cells were positive for vimentin, CD34, alpha-smooth muscle actin, muscle actin, CD34, calponin, and progesterone receptors. Ultrastructurally, many spindle cells had thin elongated cytoplasmic processes, which enveloped pseudovascular spaces containing a few collagenous fibrils, indicating fibroblastic cells. This lesion should be distinguished from other mammary fibrous and vascular lesions with which it may be confused.

Non-invasive lobular carcinoma within a fibroadenoma, a preoperatively diagnosed case.

Breast cancer within a fibroadenoma is rare and usually diagnosed postoperatively from pathological specimens. This paper reports a 54-year-old female with non-invasive carcinoma within a fibroadenoma, diagnosed preoperatively. She underwent a medical examination and mastopathy was suspected. On physical examination a mass 2 cm in diameter was palpated in the left breast. Ultrasonography showed a mass with smooth margins and uniform internal echoes, but cytology showed malignancy. Mammography showed a round mass with distinct margins and no calcification. As fibroadenoma, diagnosed by ultrasonography and mammography, and breast cancer, diagnosed by cytology, were not consistent results several core biopsies were performed. Needle biopsy showed proliferation of atypical epithelial cells; breast cancer within a fibroadenoma was diagnosed. MRI showed a circular mass with distinct, smooth margins and in a dynamic study, the mass showed irregular staining and the presence of early staining. Left lumpectomy and dissection of the left axillary lymph nodes was performed. Histological examination showed non-invasive lobular carcinoma occurring within a fibroadenoma.

Malignant phyllodes tumor in the right breast and invasive lobular carcinoma within fibroadenoma in the other: case report.

CONTEXT: The malignant variety of the phyllodes tumor is rare. The occurrence of invasive lobular carcinoma within fibroadenoma is rare as well. DESIGN: Case report. CASE REPORT: A 58-year-old black female patient was referred to the Mastology unit of the Department of Gynecology, Federal University of São Paulo / Escola Paulista de Medicina, in February 1990, presenting an ulcerated tumor in the right breast with fast growth over the preceding six months. She was a virgin, with meno-pause at the age of 45 years and had not undergone hormone replacement treatment. The physical examination showed, in her right breast, an ulcerated tumor of 20 x 30 cm which was not adher-ent to the muscle level, multilobular and with fibroelastic consistency. The axillary lymph nodes were not palpable. The left breast showed a 2 x 3 cm painless, movable nodule, with well-defined edges, and fibroelastic consistency. We performed left-breast mammography, which showed several nodules with well-defined edges, the largest being 2 x 3 cm and exhibiting rough calcification and grouped microcalcifications within it. The patient underwent a frozen biopsy that showed a malignant variant of the phyllodes tumor in the right breast and fibroadenoma in the left one. After that, we performed a total mastectomy in the right breast and an excision biopsy in the left one. Paraffin study confirmed the frozen biopsy result from the right breast, yet we observed that in the interior of the fibroadenoma that was removed on the left, there was a focal area of invasive lobular carcinoma measuring 0.4 cm. The patient then underwent a modi-fied radical mastectomy with total axillary lymphadenectomy. None of the 21 dissected lymph nodes showed evidence of metastasis. In the follow-up, the patient evolved asymptomatically and with normal physical and laboratory examination results up to July 1997.

Cytofluorometric nuclear DNA content analysis of breast tissues after frozen section diagnosis.

OBJECTIVE: To establish a suitable method for measurement of nuclear DNA content in breast tissues from frozen storage after frozen section diagnosis. STUDY DESIGN: For fundamental research, rat liver samples preserved in a deep freezer were used. Four protocols were used (1. fixation with 70% ethanol followed by naked nuclei preparation; 2. fixation with 10% neutral buffered formalin followed by naked nuclei preparation; 3. preparation for naked nuclei prior to fixation with 70% ethanol; and 4. preparation for naked nuclei prior to fixation with 70% neutral buffered formalin). For clinical research, 13 separate fresh frozen breast tissue samples were analyzed after frozen section diagnosis. One contained a malignant phyllodes tumor (MPT) consisting of 2 components, benign epithelial cells and malignant stromal cells; 3 were benign tumors containing fibroadenoma; and 9 cases were carcinomas, consisting of 5 scirrhous, 3 papillotubular and 1 mucinous. RESULTS: Protocols 1, 2 and 3 were not suitable methods for our purpose because remaining cytoplasm or cohesive nuclei were observed. In protocol 4 the cytoplasm was completely undetectable, and nuclei were suitably separated for nuclear DNA content measurement. Benign epithelial cell component nuclei presented a diploid pattern, and the malignant stromal cell component nuclei indicated a euploid pattern in MPT. All 3 cases of benign constituents in fibroadenoma showed a diploid pattern, as did the 3 carcinoma cases (1 mucinous, 1 scirrhous and 1 papillary). Four scirrhous and 2 papillary carcinomas showed an aneuploid pattern. CONCLUSION: Our findings show that it is possible to measure nuclear DNA content of human frozen storage tissues after frozen section diagnosis.

Malignant phyllodes tumor in the right breast and invasive lobular carcinoma within fibroadenoma in the other: case report

CONTEXT: The malignant variety of the phyllodes tumor is rare. The occurrence of invasive lobular carcinoma within fibroadenoma is rare as well. DESIGN: Case report. CASE REPORT: A 58-year-old black female patient was referred to the Mastology unit of the Department of Gynecology, Federal University of Sao Paulo / Escola Paulista de Medicina, in February 1990, presenting an ulcerated tumor in the right breast with fast growth over the preceding six months. She was a virgin, with meno-pause at the age of 45 years and had not undergone hormone replacement treatment. The physical examination showed, in her right breast, an ulcerated tumor of 20 x 30 cm which was not adher-ent to the muscle level, multilobular and with fibroelastic consistency. The axillary lymph nodes were not palpable. The left breast showed a 2 x 3 cm painless, movable nodule, with well-defined edges, and fibroelastic consistency. We performed left-breast mammography, which showed several nodules with well-defined edges, the largest being 2 x 3 cm and exhibiting rough calcification and grouped microcalcifications within it. The patient underwent a frozen biopsy that showed a malignant variant of the phyllodes tumor in the right breast and fibroadenoma in the left one. After that, we performed a total mastectomy in the right breast and an excision biopsy in the left one. Paraffin study confirmed the frozen biopsy result from the right breast, yet we observed that in the interior of the fibroadenoma that was removed on the left, there was a focal area of invasive lobular carcinoma measuring 0.4 cm. The patient then underwent a modi-fied radical mastectomy with total axillary lymphadenectomy. None of the 21 dissected lymph nodes showed evidence of metastasis. In the follow-up, the patient evolved asymptomatically and with normal physical and laboratory examination results up to July 1997

[The nuclear morphotypes of epithelial cells in breast diseases]. / Morfotipy iader épitelial'nykh kletok pri zabolevaniiakh molochnykh zhelez.

Staining of the nucleolar ribosome organizer with AgNO3 followed by computer analysis of images by discriminant analysis helped distinguish 6 morphological types in the cells of cytological preparations of mammary glands. Comparison of the morphometrical data and results of visual examination of cells showed that the first type included nonproliferating fibroadenoma and mastopathy, type 2 fibroadenoma and mastopathy with moderate proliferation of epithelium, type 3 fibroadenoma and mastopathy with precancer epithelial proliferation, and types 4-6 included malignant proliferating cells which are compatible with well, moderately, and poorly differentiated cancer.

[Tumor-associated changes in the buccal epithelium in human breast and thyroid pathology]. / Opukhol'-assotsiirovannye izmeneniia bukkal'nogo épiteliia pri patologii molochnoi i shchitovidnoi zhelez u cheloveka.

The estimation of the malignancy-associated changes in oral mucosa of patients with benign and malignant processes in mammary and thyroid gland is carried out on the basis of textural and densitometric indices of the interphase nuclei of the epitheliocytes with the help of the methods of mathematical statistics.

A fast non-fluorescent staining method for the detection of primed in situ synthesis products.

For the assessment of chromosomal numeral aberrations in cells, the method of choice is fluorescent in situ hybridisation (FISH) or, a newly introduced technique, oligonucleotide primed in situ hybridisation (PRINS). In the PRINS method labeled nucleotides are incorporated into newly synthesized DNA mediated through the Taq polymerase. Both PRINS and FISH reactions are visualized with a fluorescent light detection system. We present a method whereby PRINS products can be reliably and rapidly visualized by a streptavidin DAB detection system.

Diagnosis of fibroadenoma in breast fine needle aspirates devoid of typical stroma.

OBJECTIVE: To improve the accuracy of cytologic diagnosis in breast lesion aspirates that are cytologically suggestive of fibroadenoma (FA), exhibiting proliferating epithelial elements and bare nuclei but devoid of the typical stroma. STUDY DESIGN: A retrospective study was conducted. All available cases (89) that were aspirated and reported as suggestive of FA between 1981 and 1991 and were later biopsied were studied, and various morphologic criteria were analyzed in each smear. A control group consisted of 43 cases that were cytologically diagnosed as FA and confirmed histologically. RESULTS: Two criteria contributed to the cytologically suggestive diagnosis of FA. The presence of numerous "multilayered" fragments of proliferating glandular epithelium improved the positive predictive value (PPV) for FA on the suggestive smear from 67.4% to 88.3%, and the presence of numerous bare nuclei in the background improved it to 88.9%. The combined presence of both criteria improved the PPV even further, to 91.7%. CONCLUSION: When an aspirate from a breast lesion is cytologically suggestive of FA despite the absence of the typical stroma, a PPV of 91.7% can be achieved, provided that the epithelial elements include many "multilayered" fragments and that the background consists of numerous bare nuclei.

Benign pairs. A useful discriminating feature in fine needle aspirates of the breast.

OBJECTIVE: To determine the diagnostic utility of the finding of naked, bipolar nuclei in fine needle aspirates of breast lesions. STUDY DESIGN: Aspirate smears from 150 cases of histologically proven benign (77) and malignant (73) breast lesions were evaluated for the presence of stripped, bipolar nuclei in the smear background as singlets as well as closely associated pairs. RESULTS: The presence of such pairs was a more specific indicator of benign entities (present in 70% of benign lesions vs. 1% of carcinomas) when compared to single, bare nuclei alone (present in 94% of benign lesions vs. 45% of carcinomas). Large numbers of such "benign pairs" also strongly favored the diagnosis of fibroadenoma within the benign subgroup. CONCLUSION: The presence of bipolar nuclear pairs is a valuable addition to the finding of singlets in the diagnosis of benign breast lesions and their subclassification.

Study of the action of tamoxifen on the mammary gland epithelium of premenopausal patients by lysosome quantification

Tamoxifen is an antiestrogen drug widely utilized for the adjuvant hormonal treatment of breast carcinoma. Its use in the primary prophylaxis of this disease is currently being proposed. Although the drug has few side effects, its precise action on breast tissue that has not undergone neoplastic transformation has not been fully elucidated. This prospective, randomized study assessed the estrogen activity of tamoxifen on the mammary gland epithelium of premenopausal patients using a quantitative analysis of mammary epithelium lysosomes identified by the cytochemical technique of GOMORI for acid phosphatase and by light microscopy. Tamoxifen significantly increased the number of lysosomes only during the secretory phase of the menstrual cycle. We concluded that the early effect of the drug on normal mammary tissue is synergistic with the effect of estrogen during the premenopausal period.