Nuclear morphology in breast lesions: refining its assessment to improve diagnostic concordance.

AIMS: Although evaluation of nuclear morphology is important for the diagnosis and categorisation of breast lesions, the criteria used to assess nuclear atypia rely upon the subjective evaluation of several features that may result in inter- and intraobserver variation. This study aims to refine the definitions of cytonuclear features in various breast lesions. METHODS AND RESULTS: ImageJ was used to assess the nuclear morphological features including nuclear diameter, axis length, perimeter, area, circularity and roundness in 160 breast lesions comprising ductal carcinoma in situ (DCIS), invasive breast carcinoma of no special type (IBC-NST), tubular carcinoma, usual ductal hyperplasia (UDH), columnar cell change (CCC) and flat epithelial atypia (FEA). Reference cells included normal epithelial cells, red blood cells (RBCs) and lymphocytes. Reference cells showed size differences not only between normal epithelial cells and RBCs but also between RBCs in varied-sized blood vessels. Nottingham grade nuclear pleomorphism scores 1 and 3 cut-offs in IBC-NST, compared to normal epithelial cells, were < ×1.2 and > ×1.4 that of mean maximum Feret's diameter and < ×1.6 and > ×2.4 that of mean nuclear area, respectively. Nuclear morphometrics were significantly different in low-grade IBC-NST versus tubular carcinoma, low-grade DCIS versus UDH and CCC versus FEA. No differences in the nuclear features between grade-matched DCIS and IBC-NST were identified. CONCLUSION: This study provides a guide for the assessment of nuclear atypia in breast lesions, refines the comparison with reference cells and highlights the potential diagnostic value of image analysis tools in the era of digital pathology.

Quantitative tumor heterogeneity assessment on a nuclear population basis.

Immunohistochemistry Ki-67 stain is widely used for visualizing cell proliferation. The common method for scoring the proliferation is to manually select and score a hot spot. This method is time-consuming and will often not give reproducible results due to subjective selection of the hotspots and subjective scoring. An automatic hotspot detection and proliferative index scoring would be time-saving, make the determination of the Ki-67 score easier and minimize the uncertainty of the score by introducing a more objective and standardized score. Tissue Micro Array cores stained for Ki-67 and their neighbor slide stained for Pan Cytokeratin were aligned and Ki-67 positive and negative nuclei were identified inside tumor regions. A heatmap was calculated based on these and illustrates the distribution of the heterogenous response of Ki-67 positive nuclei in the tumor tissue. An automatic hot spot detection was developed and the Ki-67 score was calculated. All scores were compared with scores provided by a pathologist using linear regression models. No significant difference was found between the Ki-67 scores guided by the developed heatmap and the scores provided by a pathologist. For comparison, scores were also calculated at a random place outside the hot spot and these scores were found to be significantly different from the pathologist scores. A heatmap visualizing the heterogeneity in tumor tissue expressed by Ki-67 was developed and used for an automatic identification of hot spots in which a Ki-67 score was calculated. The Ki-67 scores did not differ significantly from scores provided by a pathologist. © 2017 International Society for Advancement of Cytometry.

Perfusion heterogeneity in breast tumors for assessment of angiogenesis.

OBJECTIVES: The purpose of this study was to investigate the perfusion heterogeneity of malignant and benign breast tumors and assay their vascular architecture changes and molecular expression, thereby evaluating the relevance between imaging and histologic characteristics of angiogenesis. METHODS: Real-time grayscale contrast-enhanced sonography was performed in 310 women with 317 breast tumors. The enhancement patterns and perfusion parameters for malignant and benign tumors were analyzed by contrast-enhanced sonography with microvascular imaging and quantitative time-intensity curve analysis. Structural characteristics were observed by light and electron microscopy. The microvessel density, vascular endothelial growth factor (VEGF) expression, and human kinase insert domain-containing receptor (KDR) expression for all tumors were assessed by immunohistochemical staining of CD31, KDR, and VEGF. RESULTS: Surgical pathologic analysis showed 163 malignant and 154 benign tumors. Significant morphologic differences, including perfusion defects, vessel distortion, vessel dilatation, and heterogeneous enhancement, were observed between the malignant and benign groups (P < .05). The mean perfusion parameters (peak intensity, ascending slope, area under the curve, and wash-out time) were greater in the malignant tumors (P < .05). There were significant differences in the peak intensity, ascending slope, area under the curve, and wash-out time between peripheral and central regions of the malignant tumors (P < .05) but none in the benign tumors. Vessels had various morphologic and distributional characteristics in the peripheral and central regions of the malignant tumors. The microvessel density and VEGF and KDR expression were significantly higher in the malignant group (P < .05), especially in the peripheral regions. CONCLUSIONS: Perfusion heterogeneity was closely associated with the tumor microvascular architecture and molecular expression. Perfusion features, especially regional morphologic and hemodynamic features, can provide valuable information for differentiating malignant from benign breast tumors.

Ranked retrieval of segmented nuclei for objective assessment of cancer gene repositioning.

BACKGROUND: Correct segmentation is critical to many applications within automated microscopy image analysis. Despite the availability of advanced segmentation algorithms, variations in cell morphology, sample preparation, and acquisition settings often lead to segmentation errors. This manuscript introduces a ranked-retrieval approach using logistic regression to automate selection of accurately segmented nuclei from a set of candidate segmentations. The methodology is validated on an application of spatial gene repositioning in breast cancer cell nuclei. Gene repositioning is analyzed in patient tissue sections by labeling sequences with fluorescence in situ hybridization (FISH), followed by measurement of the relative position of each gene from the nuclear center to the nuclear periphery. This technique requires hundreds of well-segmented nuclei per sample to achieve statistical significance. Although the tissue samples in this study contain a surplus of available nuclei, automatic identification of the well-segmented subset remains a challenging task. RESULTS: Logistic regression was applied to features extracted from candidate segmented nuclei, including nuclear shape, texture, context, and gene copy number, in order to rank objects according to the likelihood of being an accurately segmented nucleus. The method was demonstrated on a tissue microarray dataset of 43 breast cancer patients, comprising approximately 40,000 imaged nuclei in which the HES5 and FRA2 genes were labeled with FISH probes. Three trained reviewers independently classified nuclei into three classes of segmentation accuracy. In man vs. machine studies, the automated method outperformed the inter-observer agreement between reviewers, as measured by area under the receiver operating characteristic (ROC) curve. Robustness of gene position measurements to boundary inaccuracies was demonstrated by comparing 1086 manually and automatically segmented nuclei. Pearson correlation coefficients between the gene position measurements were above 0.9 (p < 0.05). A preliminary experiment was conducted to validate the ranked retrieval in a test to detect cancer. Independent manual measurement of gene positions agreed with automatic results in 21 out of 26 statistical comparisons against a pooled normal (benign) gene position distribution. CONCLUSIONS: Accurate segmentation is necessary to automate quantitative image analysis for applications such as gene repositioning. However, due to heterogeneity within images and across different applications, no segmentation algorithm provides a satisfactory solution. Automated assessment of segmentations by ranked retrieval is capable of reducing or even eliminating the need to select segmented objects by hand and represents a significant improvement over binary classification. The method can be extended to other high-throughput applications requiring accurate detection of cells or nuclei across a range of biomedical applications.

Metaplastic carcinoma of the breast: multimodality imaging and histopathologic assessment.

BACKGROUND: Metaplastic carcinomas are ductal carcinomas that display metaplastic transformation of the glandular epithelium to non-glandular mesenchymal tissue. Metaplastic carcinoma has a poorer prognosis than most other breast cancers, so the differential diagnosis is important. Although many clinical and pathologic findings have been reported, to our knowledge, few imaging findings related to metaplastic carcinoma have been reported. PURPOSE: To investigate whole-breast imaging findings, including mammography, sonography, MRI, and pathologic findings, including immunohistochemical studies of metaplastic carcinomas of the breast. MATERIAL AND METHODS: We analyzed 33 cases of metaplastic carcinoma between January 2001 and January 2011. Mammography, ultrasonography, and MRI were recorded retrospectively using the American College of Radiology (ACR) breast imaging reporting and data system (BI-RADS) lexicon. Immunohistochemical studies of estrogen receptor (ER), progesterone receptor (PR), p53, and C-erbB-2 were performed. RESULTS: The most common mammographic findings were oval shape (37%), circumscribed margin (59%), and high density (74%). The most common sonographic findings were irregular shape (59.4%), microlobulated margin (41%), complex echogenicity (81%), parallel orientation (97%), and posterior acoustic enhancement (50%). Axillary lymph node metastases were noted for 25% of the sonographic examinations. On MRI, the most common findings of margin and shape were irregularity (57% and 52.4%, respectively). High signal intensity was the most common finding on T2-weighted images (57%). Immunohistochemical profile was negative for ER (91%, 29/32) and PR (81%, 26/32). CONCLUSION: Metaplastic carcinomas might display more benign features and less axillary lymph node metastasis than IDC. High signal intensity on T2 MRI images and hormone receptor negativity would be helpful in differentiating this tumor from other breast cancers.

[Chromosome nucleolus-forming regions in assessment of neoadjuvant radiation and combined therapy for glandular breast carcinoma].

The findings of a change in the content of the main types of nucleoli and argentophilic granules involved in the structural organization, formation of active forms allow evaluation of the efficiency of radiation exposure and its combined modes during neoadjuvant therapy for glandular breast carcinoma by cytogenetic studies. A comparative study of chromosome nucleolus-forming regions before and after neoadjuvant radiotherapy and its combined modes with polychemo- and hormone therapy makes it possible to detect such aspects of therapeutic pathomorphism as the signs of tumor cell destruction and death, which appear after radiotherapy in combination with polychemotherapy.

Comparison of PathVysion and INFORM fluorescence in situ hybridization kits for assessment of HER-2/neu status in breast carcinoma.

BACKGROUND: Fluorescence in situ hybridization is advocated for precise assessment of HER-2/neu status in breast carcinoma; however, few objective data compare available kits for clinical laboratories contemplating development of the test. METHODS AND RESULTS: Thirty breast carcinomas were analyzed for HER-2/neu amplification with the PathVysion kit (Vysis, Downers Grove, IL) and INFORM kit (Ventana Medical Systems, Tucson, AZ). Each kit was evaluated for morphology, background staining, technical and interpretation time, and cost. PathVysion detected amplification in seven of 30 cases (23.3%); INFORM detected six of 30 cases (20%). A greater percentage of PathVysion cases showed good morphology and lower background staining than INFORM. Technical and interpretation times, as well as cost, were less with PathVysion than INFORM. CONCLUSION: PathVysion is superior to INFORM because it produces better morphology and less background staining and is faster and less expensive than the INFORM kit. It also includes a chromosome 17 probe that serves as an internal control and enables correction for chromosome 17 aneuploidy.

Quantitative cytological assessment of Ki67 and AgNORs using computer-digitized image analysis of four clinicopathological breast lesions.

Analysis of silver-stained proteins associated with nucleolar organiser regions (AgNORs) is proposed as a marker of cellular proliferation. This study describes the application of AgNORs and Ki67 in breast lesions. Sixty-one cases including fibroadenoma (FA), fibrocystic disease (FCD), ductal carcinoma in situ (DCIS) and invasive carcinoma (IC) were studied by image analysis to evaluate quantitative changes in AgNORs in both Ki67-positive, and Ki67-negative smears. The Ki67 index was assessed. Morphometric features of cell nuclei and AgNORs were determined by digitized computer image analysis (Prodit 5.2). The growth fraction was 5.08 for FA, 5.71 for FCD, 16.75 for DCIS and 23.26 for IC. The mean nuclear area was significantly higher in malignant cells than those of fibroadenoma and fibrocystic disease. In Ki67-positive cells the total area, long axis and number of AgNORs increased progressively across disease groups. Eccentricity of AgNORs and AgNORs: nuclear area ratios were significantly increased in malignant breast lesion in comparison with benign lesion in Ki67 positive cells. In Ki67 negative cells, the highest value of AgNORs was observed in DCIS. The AgNORs: nuclear area ratio demonstrated a statistically significant trend across the disease groups. This study demonstrates that the growth fraction, mean nuclear area and selected AgNORs features have potential for differentiating benign from malignant breast tumours.

Endothelial Tie growth factor receptor provides antigenic marker for assessment of breast cancer angiogenesis.

Breast cancer prognosis has previously been linked to the degree of tumour vascularisation. In order to establish additional markers for tumour angiogenesis, we have used monoclonal antibodies against the endothelial Tie receptor tyrosine kinase to study the degree of vascularisation of breast carcinomas and the regulation of Tie expression in the vascular endothelial cells. Antibodies were used for Tie detection and the results were correlated with other prognostic markers. Of four monoclonal antibodies directed against different epitopes of the Tie extracellular domain, two reacted against Tie in unfixed histopathological sections of breast carcinomas. One of these antibodies (clone 7e8) was specific for the endothelial cells whereas the other (clone 10f11) also reacted with basement membranes and occasional carcinoma cells. When Tie expression was studied with the antibody clone 7e8, all 27 carcinomas, two in situ carcinomas, samples of histologically normal breast tissue (n = 16) or normal skin or lymph node tissue (n = 5) showed staining. Microvessel counts were higher in carcinomas (median 14; range 3-27) than in fibrodenomas (median 10; range 5-18) or histologically normal breast tissue (median 7; range 3-15, P = 0.0006). A similar result was obtained using antibodies against the CD31 (PECAM) antigen. Microvessel counts in 7e8 staining were not significantly associated with primary tumour size, axillary nodal status, histological grade or staining for oestrogen receptor, progesterone receptor, Ki-67 proliferation marker or p53 oncoprotein.

Ductal carcinoma in situ: assessment of necrosis and nuclear morphology and their association with biological markers.

One hundred and five cases of pure ductal carcinoma in situ (DCIS) seen in the Guy's Hospital breast unit between 1975 and 1991 were reviewed. The presence and extent of necrosis and the degree of cytonuclear differentiation were assessed and the expression of p53 protein, cerbB2 protein, progesterone receptor, and a proliferation antigen KiS1, all factors reported to be of prognostic significance in invasive ductal carcinoma, was evaluated using immunohistochemical methods. A strong correlation was seen between the presence and extent of necrosis and the degree of cytonuclear differentiation and between both these morphological criteria and the biological markers as well as between the individual markers. The presence of extensive necrosis was associated with lack of cytonuclear differentiation and both were associated with a high proliferation rate, the presence of cerbB2 and p53 protein, and the absence of progesterone receptors. In cases with little or no necrosis, there was good nuclear differentiation and a strong correlation with the presence of progesterone receptor, absence of cerbB2 and p53 protein, and a low rate of proliferation.

p53 gene mutations and steroid receptor status in breast cancer. Clinicopathologic correlations and prognostic assessment.

BACKGROUND: There is increasing evidence linking development and progression of cancer to an accumulation of mutations at the genomic level. The most frequently mutated gene known to date in sporadic breast cancer appears to be the tumor suppressor gene p53. This study was designed to determine the frequency of p53 gene mutations in primary breast cancer, to correlate the presence of p53 mutations with established clinicopathologic parameters, including the estrogen receptor (ER) and progesterone receptor (PR) status, and to assess the prognostic significance of p53 mutations regarding patient survival. METHODS: We examined the p53 gene in genomic DNA samples from 192 primary breast cancers. Using denaturant gradient gel electrophoresis, the authors analyzed exons 5-9 in all tumors for mutations and performed DNA sequencing in 20 tumors to identify the exact nature of the p53 mutations. RESULTS: p53 gene alterations were identified in 43 of the 192 tumors (22%), the majority localized in exons 5 and 6. DNA sequencing showed mostly missense mutations resulting from G or C substitutions. p53 mutations were found more often in tumors of younger women (P = 0.002), Afro-American women (P = 0.05), and in tumors lacking ER (P = 0.03), PR (P = 0.04), or both (P = 0.06). There were no significant correlations with family history, tumor size, histologic grade or type, nodal status, or disease stage. The overall survival rates showed no significant difference between patients with mutant and wild-type p53 tumors. The same was true when the comparison was limited to node-negative patients or patients with ER-positive or ER-negative tumors. Finally, there was no significant difference in survival between patients with tumors harboring mutations in exons 5 and 6 versus exons 7-9. CONCLUSIONS: The results of this and other studies demonstrate a consistent relationship between ER-positive tumors and wild-type p53 on one hand and ER-negative cancers and p53 mutations on the other. Our data do not support a significant prognostic role for p53 mutations in predicting survival.

Chromatin texture measurement by Markovian analysis. Use of nuclear models to define and select texture features.

The use of nuclear grade as a prognostic indicator in breast cancer has been limited by its poor interobserver reproducibility. Automated cell classification using digital image analysis is one approach to this problem. Nuclear chromatin distribution, an important feature used in nuclear grading, can be quantitated with texture analysis. Markovian analysis is one method of analyzing texture features that is available in a commercially available image analysis system, the CAS-100. In order to select optimal Markovian features for use in nuclear grading of breast cancer, 16 nuclear models were created with computer graphics that demonstrated specific components of nuclear chromatin pattern, such as granularity, contrast, symmetry, peripheral chromatin clumping, and number and shape of nucleoli. These models were analyzed on the CAS-100 image analysis system using software capable of measuring 22 Markovian texture features at 20 levels of pixel resolution (grain). We were able to show that Markovian analysis performed well in discriminating between degrees of chromatin granularity (finely vs. coarsely clumped), amount of contrast (vesicular change), thickness of peripheral chromatin and number of nucleoli. Of the 22 Markovian features, 10 were selected as optimal for discriminating between the above chromatin patterns. Similar optimal Markovian features were found when measurements were performed on captured images of breast cancer cells. The use of these selected Markovian texture features may allow a more rational approach to the use of image analysis for cell classification.

Assessment of invasion in breast lesions using antibodies to basement membrane components and myoepithelial cells.

This paper describes immunostaining of consecutive sections from 15 cases of fibrocystic change of the breast (including 2 examples of intraductal papilloma), 4 ductal carcinomas-in-situ and 17 invasive carcinomas (4 tubular, 1 papillary, 2 lobular and 10 infiltrating ductal, NOS) with antisera to components of the basement membrane (BM), type IV collagen and laminin, and with the muscle antibodies actin and muscle-specific actin. A simple digestion technique was developed to improve the clarity of BM staining with these antibodies. The BM stains facilitated identification of small invasive foci through breaks in the BM in 2 of the cases which had been reported as pure intraductal carcinoma. Tubular carcinomas were surrounded by abnormal, fragmented, and focally discontinuous BM, a feature which could be used to distinguish this well-differentiated breast carcinoma sub-type from sclerosing adenosis, in which individual acini were invariably surrounded by a continuous BM. BM staining emphasized the fibrovascular core of intraductal papillomas, whereas the BM layer was absent in intraductal, cytologically malignant, papillary projections. Similarly, myoepithelial cells, stained with antisera to muscle actins, were identified in a continuous layer surrounding benign epithelial proliferations. These immunohistochemical staining techniques may thus assist the diagnostic histopathologist in differentiating between benign epithelial proliferations of the breast and well-differentiated invasive breast carcinoma, and in identifying foci of microinvasive carcinoma.

Immunohistochemical assessment of ER-P31, a mouse anti-oestrogen receptor protein monoclonal antibody in human breast cancers: comparison with ER-ICA (Abbott) and radioligand assays.

A new mouse anti-oestrogen receptor (ER) monoclonal antibody, ER-P31, has been developed. Comparisons of immunohistochemical detection of ER in human breast cancers using ER-P31 with anti-ER from ER-immunocytochemical assay (ER-ICA; Abbott Diagnostic Division) and radioligand dextran-coated charcoal (DCC) assays were carried out. A total of 63 breast cancers, both ER-negative and -positive, were tested. A highly significant degree of correlation between immunostaining for ER-P31 and both ER-ICA and DCC assays was obtained. It is hoped that once ER-P31 is widely available commercially, determination of ER status in breast cancers should be routinely and economically available to all women with breast cancer. Moreover, with the introduction and implementation of a screening programme for detecting breast cancers, immunocytochemical determination of ER status in unequivocal and equivocal positive fine-needle aspirates of breast lesions can be readily performed.

Flow cytometric analysis of human breast tumors and assessment of in vitro chemosensitivity by clonogenic assays.

We report a double-agar clonogenic system adapted to human breast cancer. We optimized the conditions for cell growth and clonogenicity with respect to hormones (insulin, estradiol, progesterone) and components of the extracellular matrix (collagen, laminin and fibronectin). Using our experimental improvements, 67% of the breast tumor samples received were grown successfully. Tests on 21 tumors with three agents: Doxorubicin, Methotrexate and 5-Fluorouracil permit objective discrimination of the in vitro pharmacosensitivity of human breast tumors. Flow cytometric analysis reveal that 64% of the tumors were diploid and 36% were aneuploid. The aneuploid tumors grew better in the double agar layer system used for the clonogenic assay. The diploid tumors were especially rich in estrogen (ER+) and progesterone (PR+) receptors whereas the aneuploid tumors were mostly estrogen and progesterone receptors negative (ER-/PR-). Finally, we noted no difference in drug responsiveness depending on the tumor ploidy and steroid receptor content.

Morphometrical assessment of mean nuclear area in breast cancer in comparison with that of lymph node metastases.

Morphometrical measurements of nuclear area and form factors are carried out by means of a semiautomated image analyzer on 90 cases of ductal breast cancers and on lymph node tumor deposits. The value of the mean nuclear area in lymph node metastases is significantly higher than in primary tumors regardless of the size of the tumor. Since the value of mean nuclear area is also significantly higher in primary tumors with lymph node involvement than in those without lymph node involvement it is possible to assume that primary tumors with large-nucleus areas are more liable to invade lymph nodes. No significant differences are noted as far as the form factors are concerned.

The assessment of histological differentiation in breast cancer.

Morphological tumour differentiation has been shown in numerous studies to give a good prediction of prognosis in breast cancer. In the Nottingham/Tenovus study, tumour differentiation (histological grade) is assessed as part of a routine analysis of potential prognostic factors. Three features are analysed, the degree of tubule formation, variation in the size and shape of nuclei and mitotic rate. Each feature is given a score of 1-3 and grade is assigned as follows: Grade I, well differentiated, 3-5 points; Grade II, moderately differentiated, 6-7 points; Grade III, poorly differentiated, 8-9 points. Histological grade has been assessed in 625 patients with primary breast carcinoma and there is a strong correlation with prognosis; patients with well differentiated tumours have a significantly better survival than those with poorly differentiated tumours (P less than 0.0005). Grade forms an important part of the Nottingham/Tenovus prognostic index together with tumour size and lymph node stage. It is suggested that factors such as these should be used to stratify patients for appropriate therapy.