BMP-2 Variants in Breast Epithelial to Mesenchymal Transition and Microcalcifications Origin.

This study aims to investigate the possible different roles of the BMP-2 variants, cytoplasmic and nuclear variant, in both epithelial to mesenchymal transition and in microcalcifications origin in human breast cancers. To this end, the in situ expression of cytoplasmic and nuclear BMP-2 was associated with the expression of the main epithelial to mesenchymal transition biomarkers (e-cadherin and vimentin) and molecules involved in bone metabolisms (RUNX2, RANKL, SDF-1) by immunohistochemistry. In addition, the expression of cytoplasmic and nuclear BMP-2 was associated with the presence of microcalcifications. Our data showed a significant association among the number of cytoplasmic BMP-2-positive cells and the number of both vimentin (positive association) and e-cadherin (negative association) positive breast cells. Conversely, no associations were found concerning the nuclear BMP-2-positive breast cells. Surprisingly, the opposite result was obtained by analyzing the variants of BMP-2 and both the expression of RANKL and SDF-1 and the presence of microcalcifications. Specifically, the presence of microcalcifications was related to the expression of nuclear BMP-2 variant rather than the cytoplasmic one, as well as a strong association between the number of nuclear BMP-2 and the expression of the main breast osteoblast-like cells (BOLCs) biomarkers. To further corroborate these data, an in vitro experiment for demonstrating the co-expression of nBMP-2 and RANKL or vimentin or SDF-1 in breast cancer cells that acquire the capability to produce microcalcifications was developed. These investigations confirmed the association between the nBMP-2 expression and both RANKL and SDF-1. The data supports the idea that whilst cytoplasmic BMP-2 can be involved in epithelial to mesenchymal transition phenomenon, the nuclear variant is related to the essential mechanisms for the formation of breast microcalcifications. In conclusion, from these experimental and translational perspectives, the complexity of BMP-2 signaling will require a detailed understanding of the involvement of specific BMP-2 variants in breast cancers.

Toward the first definition of taste rosea in female breasts: histological analysis / La primera definición del sabor rosea en mamas femeninas: análisis histológico

Although various neuropsychochemical theories have been established about why breastfeeding mothers feel hedonic sensation, the underlying neural mechanism has not been adequately clarified. We aimed to investigate if there is hedonic sensation-initiated taste-bud like structures stimulated by sugars in the milk-secreting lactiferous ducts of mammary glands of breastfeeding female rats. In this study, twenty-two female rats were chosen which six of the virgin (n=6), six of pregnant (n=6) and ten of breastfeeding (n=10). We examined lactiferous ducts/nipples of mammary glands of all animals. They were sacrificed following intracardiac formalin injection, and their breast tissues were removed with covering tissues and fixed with 10 % of formalin solution. After current histological procedures, the tissues were examined by light microscope to assess taste-bud like structures, and their numerical densities were calculated by using stereological methods. Results were analyzed statistically. Taste-buds like structures with neuron-like appendages at the apical ends were discovered in lactiferous ducts. The taste rosea numbers were estimated as 3±1/mm3 in virgins, 167±27/mm3 in pregnant and 375±63/mm3 in breastfeeding animals. The taste rosea numbers were greater in breastfeeding rats than those of virgins and pregnant rats. They named as taste rosea resembling flower bucket which has not been mentioned in the literature so far.

Existen varias teorías neuropsicoquímicas, referente a la sensación hedónica que sienten las mujeres al amamantar, y el mecanismo neural subyacente. No obstante, estas aún no se aclaran adecuadamente. El objetivo de este estudio, fue investigar si existen estructuras hedónicas iniciadas por la sensación gustativa estimuladas por los azúcares en los conductos mamarios secretores de leche, de las glándulas mamarias de las ratas durante el período de lactancia. En este estudio, se eligieron 22 ratas hembras, seis de estas no preñadas como grupo control, seis preñadas y diez en período de lactancia. Examinamos los conductos lactíferos / pezones de las glándulas mamarias de los tres grupos. Los animales fueron sacrificados por medio de inyección intracardíaca de formalina. El tejido mamario se fijó en solución de formalina al 10 %. La muestras histólogicas fueron examinadas a través microscopía óptica con la finalidad de evaluar estructuras con características morfológicas similares a las papilas gustativas. Su densidad de número se calculó utilizando métodos estereológicos. Los resultados fueron analizados estadísticamente. En los conductos mamarios se observaron dos estructuras con con características morfológicas tipo papilas gustativas con apéndices neuronales en los extremos apicales. Los números se estimaron en 3±1/mm3 en el grupo control, 167±27/mm3 en gestantes y 375±63/mm3 en animales lactantes. El número de estructuras características morfológicas similares a las papilas gustativas fue mayor en las ratas amamantando que en el grupo control y que en las ratas preñadas. Conocido como sabor rosea debido a que se asemeja a un ramo de flores, lo que hasta ahora no se ha mencionado en la literatura.

Feasibility of abbreviated magnetic resonance imaging (AB-MRI) screening in women with a personal history (PH) of breast cancer.

PURPOSE: To investigate the feasibility of abbreviated magnetic resonance imaging (AB-MRI) in women with a personal history (PH) of breast cancer as a screening tool. MATERIALS AND METHODS: We retrospectively reviewed 1880 screening AB-MRIs in 763 women with a PH of breast cancer (median age, 55 years; range, 23-89 years) between October 2015 and October 2016. The total acquisition times of AB-MRI were 8.3 min and 2.8 min with and without T2-weighted imaging, respectively. The tissue diagnosis or one-year follow-up status was used as the reference standard. The characteristics of tumor recurrences detected on AB-MRI screenings were analyzed. The cancer detection rates (CDRs) and additional CDRs for the 1st round and overall rounds of AB-MRI screening were calculated. The recall rate, sensitivity, specificity, positive predictive values for recall (PPV1) and biopsy (PPV3) for the 1st round of AB-MRI screening were calculated. The diagnostic performance of the combination of mammography and ultrasonography was compared with that of AB-MRI by receiver operating characteristic (ROC) curve analysis. RESULTS: Fifteen of a total of 21 recurrences were detected on the 1st round of AB-MRI screening: 93.3% were node-negative T1 tumors (median tumor size, 1.02 cm; range, 0.1-2 cm) or Tis; 66.7% were high-grade tumors; 8 of these 15 were mammographically and ultrasonographically occult. The CDR and additional CDR for the 1st round of AB-MRI screening were 0.019 and 0.010 per woman, respectively. The sensitivity, specificity, recall rate, PPV1 and PPV3 for the 1st round of AB-MRI screening were 100%, 96.0%, 14.3%, 13.8% and 58.3%, respectively. For detecting secondary cancer, AB-MRI showed a higher sensitivity and PPV than the combination of mammography and ultrasonography (95.2%, 57.1% vs 47.6%, 38.5%). The area under the ROC curve was higher for AB-MRI (0.966; 95% CI: 0.951-0.978) than the combination of mammography and ultrasonography (0.727; 95% CI: 0.694-0.759) (P<0.0001). CONCLUSION: AB-MRI improved cancer detection with a high specificity, sensitivity and PPV in women with a PH of breast cancer. AB-MRI could be a useful screening tool for detecting secondary cancer considering its high diagnostic performance and short examination time.

Structural Studies of Epithelial Mesenchymal Transition Breast Tissues.

At the supramolecular level, the proliferation of invasive ductal carcinoma through breast tissue is beyond the range of standard histopathology identification. Using synchrotron small angle x-ray scattering (SAXS) techniques, determining nanometer scale structural changes in breast tissue has been demonstrated to allow discrimination between different tissue types. From a total of 22 patients undergoing symptomatic investigations, different category breast tissue samples were obtained in use of surgically removed tissue, including non-lesional, benign and malignant tumour. Structural components of the tissues were examined at momentum transfer values between q = 0.2 nm-1 and 1.5 nm-1. From the SAXS patterns, axial d-spacing and diffuse scattering intensity were observed to provide the greatest discrimination between the various tissue types, specifically in regard to the epithelial mesenchymal transition (EMT) structural component in malignant tissue. In non-lesional tissue the axial period of collagen is within the range 63.6-63.7 nm (formalin fixed paraffin embedded (FFPE) dewaxed) and 63.4 (formalin fixed), being 0.9 nm smaller than in EMT cancer-invaded regions. The overall intensity of scattering from cancerous regions is a degree of magnitude greater in cancer-invaded regions. Present work has found that the d-spacing of the EMT positive breast cancer tissue (FFPE (dewaxed)) is within the range 64.5-64.7 nm corresponding to the 9th and 10th order peaks. Of particular note in regard to formalin fixation of samples is that no alteration is observed to occur in the relative differences in collagen d-spacing between non-lesional and malignant tissues. This is a matter of great importance given that preserved-sample and also retrospective study of samples is greatly facilitated by formalin fixation. Present results indicate that as aids in tissue diagnosis SAXS is capable of distinguishing areas of invasion by disease as well as delivering further information at the supramolecular level.

Characterization of breast tissue permeability for detection of vascular breast tumors: An in vitro study.

The biological tissue could be considered as a porous matrix filled with the interstitial fluid. The tumoral tissue has a different permeability from the healthy tissue. With regard to this knowledge, the main objective of the present study is to study the tissue permeability as a diagnostic parameter for the detection of breast cancer. To this end, the healthy and the cancerous specimens of the breast tissue are taken from 17 female cases. All cancerous tumors are in the vascular phase of the growth. The samples undergo a uniaxial compression test by a robotic system while the strain rate is set to remain unchanged. Using the stress and the strain rate data, the strain-dependent permeability is determined, which is an exponential function of the strain level. The permeability function is identified by the initial permeability at the zero compressive strain and a material constant. Results show that the initial permeability of the healthy breast tissue is significantly different from the corresponding value for the cancerous tissue. For all cancerous samples, the permeability is less than the healthy tissue samples; as 40-70% reduction in the initial permeability is observed compared to the healthy breast tissue. The evaluation of the permeability between the healthy and the cancerous specimens is accompanied by the biopsy reports and observing structural images of the specimens using environmental scanning electron microscope. Based on the results, the permeability is suggested to have a diagnostic application for the detection of vascular breast tumors.

Core needle biopsy of benign, borderline and in-situ problematic lesions of the breast: Diagnosis, differential diagnosis and immunohistochemistry.

Core needle biopsy (CNB) is the most common sampling technique for the histologic evaluation of breast abnormalities. Diagnosing benign proliferative, borderline and some in-situ lesions in CNB is challenging and subject to a significant degree of interobserver variability. In addition, due to the inherent limitations of CNB, "upgrading" to a more significant pathology at excision is an important consideration for some lesions. Pathologists carry a major responsibility in patient diagnosis, risk stratification and management. Familiarity with the histologic features and the clinical significance of these common and problematic lesions encountered in CNB is necessary for adequate treatment and patient follow-up. This review will focus on benign, atypical and in-situ epithelial proliferations, papillary lesions, radial sclerosing lesions, adenosis and cellular fibroepithelial lesions. Highlights of histologic features, useful strategies for accurate diagnosis, basic immunohistochemistry and management will be presented.

High-resolution 3D imaging of fixed and cleared organoids.

In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.

Expression of Luminal Progenitor Marker CD117 in the Human Breast Gland.

CD117 is a putative marker of luminal progenitor cells in the human breast. However, so far mapping the expression pattern of CD117 within the normal gland has not been reported. Here, we examined the anatomical distribution of CD117-expressing cells in lobular and ductal structures by immunohistochemistry. The presence of CD117-positive luminal cells could be divided into three distinct patterns: (1) contiguous, with coherent positive cells and rare negative cells interspaced; (2) patched, with a roughly equal frequency of positive and negative cells distributed focally; or (3) scattered, with few or no positive cells in the structure. Generally, a patched or scattered expression pattern was more frequent in lobules compared with ducts. Furthermore, an age-correlated increase in heterogeneity was observed. When comparing women below and above 21 years of age this heterogeneity was evident for both lobules and ducts. Although CD117-expression was generally segregated from luminal-lineage transcription factor GATA3-positive cells, some did co-express both markers. Finally, co-staining with Ki-67 revealed that a prominent part of cycling cells belonged to the CD117-positive population. Together these data demonstrate the presence of a CD117-expressing progenitor compartment with the capacity to replenish the luminal lineage of the breast gland.

The significance of tumour microarchitectural features in breast cancer prognosis: a digital image analysis.

BACKGROUND: As only a minor portion of the information present in histological sections is accessible by eye, recognition and quantification of complex patterns and relationships among constituents relies on digital image analysis. In this study, our working hypothesis was that, with the application of digital image analysis technology, visually unquantifiable breast cancer microarchitectural features can be rigorously assessed and tested as prognostic parameters for invasive breast carcinoma of no special type. METHODS: Digital image analysis was performed using public domain software (ImageJ) on tissue microarrays from a cohort of 696 patients, and validated with a commercial platform (Visiopharm). Quantified features included elements defining tumour microarchitecture, with emphasis on the extent of tumour-stroma interface. The differential prognostic impact of tumour nest microarchitecture in the four immunohistochemical surrogates for molecular classification was analysed. Prognostic parameters included axillary lymph node status, breast cancer-specific survival, and time to distant metastasis. Associations of each feature with prognostic parameters were assessed using logistic regression and Cox proportional models adjusting for age at diagnosis, grade, and tumour size. RESULTS: An arrangement in numerous small nests was associated with axillary lymph node involvement. The association was stronger in luminal tumours (odds ratio (OR) = 1.39, p = 0.003 for a 1-SD increase in nest number, OR = 0.75, p = 0.006 for mean nest area). Nest number was also associated with survival (hazard ratio (HR) = 1.15, p = 0.027), but total nest perimeter was the parameter most significantly associated with survival in luminal tumours (HR = 1.26, p = 0.005). In the relatively small cohort of triple-negative tumours, mean circularity showed association with time to distant metastasis (HR = 1.71, p = 0.027) and survival (HR = 1.8, p = 0.02). CONCLUSIONS: We propose that tumour arrangement in few large nests indicates a decreased metastatic potential. By contrast, organisation in numerous small nests provides the tumour with increased metastatic potential to regional lymph nodes. An outstretched pattern in small nests bestows tumours with a tendency for decreased breast cancer-specific survival. Although further validation studies are required before the argument for routine quantification of microarchitectural features is established, our approach is consistent with the demand for cost-effective methods for triaging breast cancer patients that are more likely to benefit from chemotherapy.

Correlative imaging reveals physiochemical heterogeneity of microcalcifications in human breast carcinomas.

Microcalcifications (MCs) are routinely used to detect breast cancer in mammography. Little is known, however, about their materials properties and associated organic matrix, or their correlation to breast cancer prognosis. We combine histopathology, Raman microscopy, and electron microscopy to image MCs within snap-frozen human breast tissue and generate micron-scale resolution correlative maps of crystalline phase, trace metals, particle morphology, and organic matrix chemical signatures within high grade ductal carcinoma in situ (DCIS) and invasive cancer. We reveal the heterogeneity of mineral-matrix pairings, including punctate apatitic particles (<2 µm) with associated trace elements (e.g., F, Na, and unexpectedly Al) distributed within the necrotic cores of DCIS, and both apatite and spheroidal whitlockite particles in invasive cancer within a matrix containing spectroscopic signatures of collagen, non-collagen proteins, cholesterol, carotenoids, and DNA. Among the three DCIS samples, we identify key similarities in MC morphology and distribution, supporting a dystrophic mineralization pathway. This multimodal methodology lays the groundwork for establishing MC heterogeneity in the context of breast cancer biology, and could dramatically improve current prognostic models.

[Clinicopathologic study of infiltrating epitheliosis of the breast].

Objective: To evaluate the morphological and immunohistochemical features of infiltrating epitheliosis and its differential diagnosis. Methods: Nine consultation and routine cases of infiltrating epitheliosis diagnosed from January 2015 to December 2016 in Fudan University Shanghai Cancer Center were collected. All tissues were formalin-fixed paraffin-embedded and routinely HE stained. The HE slides were reviewed. Immunohistochemical staining of CKpan, CK7, CK19, CK5/6, CK14, p63, SMMHC, Calponin, ER, PR, HER2, Ki-67 and S-100 protein was performed using Ventana BenchMark automated immunostainer. Results: The morphological features of infiltrating epitheliosis included: (1) Florid proliferation of epithelial cells forming solid nests or papillary, glandular and cord-like pattern. The proliferative cells possessed nuclei of varying size and shape without atypia. (2) The stroma was altered, showing varying degrees of fibrosis or sclerosis. (3) The proliferative epithelial nests might flow into the spaces within small ducts and lobules at the periphery of the lesion, resulting in pseudo-infiltration. Immunohistochemically, infiltrating epitheliosis was non-uniformly positive for ER/PR, and was positive for high molecular weight CK5/6 and CK14. Myoepithelial markers p63, SMMHC and Calponin demonstrated intact, partial or entire loss of myoepithelial cells around the epithelial nests. The loss of myoepithelial markers staining was more frequent at the periphery of the lesion. The most important differential diagnoses included invasive ductal carcinoma, ductal carcinoma in situ (DCIS), and low grade adenosquamous carcinoma, etc. Conclusions: Infiltrating epitheliosis is an important pseudo-infiltrating lesion. The lack of atypia, non-uniform ER/PR expression, positivity for high molecular weight cytokeratins, and the intact to partial to entire loss of myoepithelial markers around the proliferating cell nests are the key points to differentiate it from invasive carcinomas and DCIS.

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.

Molecular analysis of BRCA1 and BRCA2 genes by next generation sequencing and ultrastructural aspects of breast tumor tissue.

In this paper, we focus our interest on the dynamics alterations of the tumor-stroma interface at the ultrastructural level and to detect BRCA1 and BRCA2 mutations using next generation sequencing (NGS) of breast tumor tissue. Electron microscopic investigation revealed some peculiar infrastructural alterations of the tumor cells per se as well as of the tumor-stroma interface: invadopodia, shedding microvesicles, altered morphology and reduced number of telocytes, different abnormalities of the microvasculature. Tumor suppressor genes BRCA1 and BRCA2 are the genes with most hereditary predisposition to breast and ovarian cancer. An early identification of mutation within these genes is essential for determining classification and therapeutic approach to patients. Genetic tests used to determine mutations in BRCA1 and BRCA2 genes are laborious analysis methods which include, among others, NGS. We analyzed a total of eight samples, in which genomic DNA was amplified using Ion AmpliSeq panel BRCA1 and BRCA2. DNA libraries were created, amplified and sequenced with Ion Torrent Personal Genome Machine. The bio-information data obtained allow us to detect all known pathogenic mutation and uncertain polymorphisms.

Elemental vs. phase composition of breast calcifications.

Despite the importance of calcifications in early detection of breast cancer, and their suggested role in modulating breast cancer cell behaviour, very little detail is known about their chemical composition or how this relates to pathology. We measured the elemental composition of calcifications contained within histological sections of breast tissue biopsies, and related this to both crystallographic parameters measured previously in the same specimens, and to the histopathology report. The Ca:P ratio is of particular interest since this theoretically has potential as a non-invasive aid to diagnosis; this was found to lie in a narrow range similar to bone, with no significant difference between benign and malignant. The Mg:Ca ratio is also of interest due to the observed association of magnesium whitlockite with malignancy. The initially surprising inverse correlation found between whitlockite fraction and magnesium concentration can be explained by the location of the magnesium in calcified tissue. Sodium was also measured, and we discovered a substantial and significant difference in Na:Ca ratio in the apatite phase between benign and malignant specimens. This has potential for revealing malignant changes in the vicinity of a core needle biopsy.

The effect of chemotherapy on the mammographic appearance of breast cancer and correlation with histopathology.

OBJECTIVE: To document the mammographic changes after neoadjuvant chemotherapy with histopathological correlation, to calculate the accuracy of mammography (MG) in predicting residual tumour size and to measure the interobserver agreement in reading mammograms. METHODS: In 446 consecutive cases, the pre- and post-chemotherapy mammograms were retrospectively evaluated by two blinded observers, and consensus findings were compared with reference standard of surgical specimen. The accuracy of MG in predicting residual tumour size was calculated. Kappa statistics were calculated for measuring the interobserver agreement for reading mammograms. The sensitivity, specificity, positive-predictive value and negative-predictive value for the prediction of residual disease were calculated. RESULTS: The most common primary abnormalities were mass lesions without and with microcalcifications. After chemotherapy, there was decrease in size of most (95.1%) of the measurable masses, with decrease in the mean tumour size from 4.1 to 2.5 cm. The density of the tumour decreased in 66.6% (241/362) cases with residual disease. There was almost perfect interobserver agreement for describing the primary abnormality in the pre- as well as post-chemotherapy mammograms (k = 0.87 and 0.81, respectively) with substantial agreement for measurement of the mass lesions before and after chemotherapy (k = 0.69 and 0.68, respectively). MG showed accuracy of 60.0%, sensitivity of 94.4%, specificity of 50.0%, positive-predictive value of 91.3% and negative-predictive value of 61.8%. CONCLUSION: MG remains a highly sensitive and reproducible investigation for the assessment of residual disease after chemotherapy. ADVANCES IN KNOWLEDGE: There is substantial interobserver agreement in characterizing and measuring breast tumours on mammograms.

A case study on medical image enhancement based on II curvelet.

A method is presented in this paper for medical image enhancement based on II curvelet. After the wavelet decomposition of medical image, we continue to break down the high-frequency sub-images, in order to get more detailed information. We have also designed the corresponding gain weight function for the edge enhancement of low-frequency sub-images, using II curvelet to extract the edge information, which has advantages over the normal curvelet transform. This method has overcome the disadvantages of the present medical image enhancement algorithm based on wavelet theory, for instance, (1) most of the present methods only decompose the low-frequency sub-images to get detailed information; (2) they can not well extract the tumors or other large areas of edge information of medical images. In simulation experiments, we enhanced the mammography X-ray images of breast provided by Heilongjiang Provincial Tumor Hospital, and compared our algorithm with several traditional image enhancement methods. The results shows that with our enhancement algorithm based on II curvelet, the textures and edges of the image can be reflected clearly, and the calcifications in the image are independent, at the same time, this method has the superiority when enhancing the images which have been added noise to.