Automated image morphometry of lobular breast carcinoma

OBJECTIVE: To analyze the role of automated image morphometry (AIM) in distinguishing infiltrating lobular carcinoma (ILC) of the breast from benign, borderline and infiltrating ductal carcinoma (IDC). STUDY DESIGN: Only histopathologically proven lobular carcinoma, ductal carcinoma, borderline lesions and benign breast lesions were selected for the study. There were 19 cases of ILC and 30 cases of IDC, 20 cases of benign lesions (fibroadenoma, 18; fibrocystic disease, 1; and fibroadenosis, 1); 10 cases were borderline lesions (mild epithelial hyperplasia, 3; moderate epithelial hyperplasia, 2; florid epithelial hyperplasia 4; intraductal papillary carcinoma, 1). In all cases hematoxylin and eosin-stained slides were used for AIM. At least 100 cells from each case were subjected to analysis randomly with an image cytometer with Leica Quantimet 600 software (Cambridge, England). Nuclear area, diameter, perimeter, convex perimeter, convex area and roundness were measured in each case with random, unbiased selection of cells and 40 x objectives (one pixel = 0.46 microm). AIM data on the cases were analyzed in relation to final cytologic diagnosis. RESULTS: All the nuclear morphometric features of ILC were much lower than those of IDC and borderline lesions, whereas nuclear morphometric data on ILC were only marginally more than those on benign cases. ANOVA showed that mophometric data were significant (P < .05) in all the variables between ILC and IDC. However, there was no significant difference between ILC, and borderline and benign cases. CONCLUSION: Image morphometry may be useful in distinguishing ILC from IDC on cytologic smears. However, morphometric data may not be helpful in distinguishing benign and borderline lesions from ILC.     

Robinson cytologic grading of invasive ductal breast carcinoma: correlation with histologic grading and regional lymph node metastasis

OBJECTIVE: To evaluate the importance of cytologic grading of breast carcinoma and its association with histologic grading and the existence of axillary lymph node metastasis. STUDY DESIGN: Aspirates and surgical samples from 100 patients with invasive ductal breast carcinoma not otherwise specified were studied. In 50 patients, > or = 1 metastatic nodes were identified. The cytologic grade was evaluated using the Robinson method and the histologic grade using the Elston modification of the Bloom-Richardson method. A study was undertaken to establish the association between histologic and cytologic grades and to compare the various parameters used to evaluate cytologic grade with the presence of axillary node metastasis. RESULTS: A statistically significant association was observed between cytologic and histologic grades (p < 0.0005) and between cytologic grade and presence of axillary metastasis (p < 0.0005). Similarly, cell dissociation (p < 0.0005), cell uniformity (p = 0.0010) and the appearance of nuclear margins (p < 0.0005) all displayed a positive correlation with regional metastasis. CONCLUSION: Cytologic grade may provide relevant information on the aggressiveness of invasive ductal breast carcinoma and could be a useful parameter to take into consideration when selecting neoadjuvant therapy.     

Nuclear image morphometry and cytologic grade of breast carcinoma

OBJECTIVE: To correlate visual cytologic grade with automated nuclear morphometry of carcinoma of the breast. STUDY DESIGN: We randomly selected 24 histologically proven infiltrating ductal carcinomas of the breast and 10 benign breast lesions (fibroadenoma). Hematoxylin-eosin-stained fine needle aspiration cytology (FNAC) smears were selected for both cytologic grade and automated image morphometry. The same hematoxylin-eosin-stained FNAC smears were studied for area, convex area, standard deviation of nuclear area, diameter, perimeter and convex perimeters of nucleus. At least 100 cells from each case were measured with an image cytometer. RESULTS: Mean nuclear area, standard deviation of nuclear area, nuclear diameter, convex area, convex perimeter and perimeter were significantly increased from benign versus grade 1 carcinomas and grade 1 versus grade 2 and 3 carcinomas (one way ANOVA test). However, there was no significant difference in grade 2 versus grade 3 carcinomas. CONCLUSION: Automated image cytometry rapidly and successfully measures various nuclear parameters. The measurement of various nuclear parameters would be helpful in future applications of automated diagnosis and grading of breast carcinomas from cytologic material.     

Fine needle aspiration cytology of ductal breast carcinoma with neuroendocrine differentiation. Review of eight cases with histologic correlation

OBJECTIVE: To describe the fine needle aspiration cytology findings of ductal breast carcinoma with neuroendocrine differentiation and correlate them with the histologic appearance. STUDY DESIGN: We reviewed the cytologic features of eight cases of ductal carcinoma with neuroendocrine differentiation in the files of Pamela Youde Nethersole Eastern Hospital during the three-year period 1998-2000. Immunohistochemical study for neuroendocrine markers was performed, with ultrastructural correlation. RESULTS: All cases showed similar cytologic features. The smears were of moderate to high cellularity with predominantly dispersed or loosely cohesive tumor cells. The carcinoma cells were mostly of low cytologic grade. They possessed round and relatively uniform, eccentric nuclei; finely stippled chromatin; sometimes small, distinct nucleoli; and discrete cell borders. Abundant eosinophilic and focally granular cytoplasm was a common finding. In some of the cases there was accentuation of staining in the paranuclear region; it correlated with aggregates of dense core neurosecretory granules seen ultrastructurally. Mucoid substance was seen in the background in some of the aspirates. Histologic examination of the tumors showed invasive ductal carcinoma with an organoid growth pattern and sometimes mucinous component. The neuroendocrine differentiation was confirmed immunohistochemically. CONCLUSION: Although this subtype of ductal carcinoma probably carries no significant prognostic value per se, it has distinct cytologic features, rendering preoperative diagnosis possible. Recognition of this entity is important in order to avoid the misdiagnosis of neuroendocrine tumor metastatic to the breast.     

Nuclear morphometry and estrogen receptors in infiltrating ductal carcinoma of the breast

In this study ten cases of breast infiltrating ductal carcinoma have been considered. In all of them the content of ER has been evaluated by using monoclonal antibodies. Five of them were ER positive and five were ER negative. For the morphometric study ten nuclei of each case have been considered. By using the S.A.M. (Shape Analytical Morphometry) work-station an analytical study of the nuclear shape was performed. The first step was the extraction of fundamental shape which describes the basic shape of original contour without its irregularities. It was obtained by using two parametric equations. The second step was the evaluation of shape asymmetry by S.A.E. (Shape Asymmetry Evaluator). Finally the contour irregularities were evaluated by Fourier analysis. Along with analytical parameters, dimensions (area, perimeter and maximum diameter) were considered too. All obtained data were submitted to univariate statistical analysis (Student's T test) to compare the two groups (ER positive and ER negative tumors). Area, perimeter and maximum diameter were significatively greater in ER negative cases while analytical parameters were not discriminant between the two groups.     

FNA of breast fibroadenoma: observer variability and review of cytomorphology with cytohistological correlation

OBJECTIVE: To determine the observer variability in reporting fibroadenoma of the breast by fine needle aspiration (FNA) and to review the cytomorphological features of the lesion with cytohistological correlation. METHODS: Retrospective analysis of FNA smears from 110 cases diagnosed as fibroadenoma of which surgical pathology follow-up was available in 33. Two pathologists were asked to categorize smears from 67 cases of breast lesions while blinded to the clinical finding as fibroadenoma, epithelial hyperplasia (usual and atypical) and malignant. All fibroadenoma (33) and cancer (15) cases were biopsy-proven. The same set of slides was re-circulated to one of the pathologists, and his first and second round results were compared. RESULTS: Pre-review cytohistological correlation was attained in 32 of 33 cases of fibroadenoma (97%). The overall agreement between the two observers was 87% [Kappa = 0.74, 95% confidence interval (CI) 0.72-0.76]. Cytohistological correlation was achieved in 26 of 33 (79%) cases. Intra-observer agreement was 91% (Kappa = 0.82, 95% CI 0.89-0.93) with cytohistological correlation in 29 of 33 (87%) cases. Causes of diagnostic errors included marked dissociation, pleomorphism, poorly cellular smears from hyalinized fibrodenoma, lacational changes and apocrine metaplasia with cystic changes. Multinucleated giant cells were frequently encountered in FNA smears from fibroadenoma (31.8%), but in none of the lumpectomy specimens. Their histiocytic nature was suggested by immunohistochemistry. CONCLUSION: FNA was a highly sensitive method for the diagnosis of fibroadenoma. Current cytological criteria were reliable and gave high inter- and intra-observer reproducibility.     

Computerized image analysis of Ki-67 in ductal breast carcinoma in situ.

OBJECTIVE: To develop and determine the staining protocols and computerized image analysis methods that are the most effective combination for performing quantitative analysis of Ki-67. STUDY DESIGN: We compared conventional bright-field light microscopy and refractive optical enhancement methods in combination with various immunodetection and filter enhancement methods, including immunogold in combination with epipolarization refractive optics and enzymatic conversion of chromogenic substrates in combination with optical filter enhancement. Initial Ki-67 tests were performed on lymph node tissues and cultured human breast cells and then applied to 200 ductal carcinoma in situ (DCIS) samples. DCIS acini were digitally acquired, and a region of interest was manually outlined in each one with a digital stylus to include only the cellular component; then the Ki-67 staining index was quantified by segmentation analysis. RESULTS: Although combining epipolarization analysis with immunohistogold staining was the most sensitive detection method, nonspecific binding was too high. The streptavidin-horseradish-peroxidase enzymatic conversion of 3,3'-diaminobenzidine (DAB) in combination with optical enhancement filters was the most effective method tested. Ki-67 stain was associated with dense fibrillar structures of the nucleoli in the less intensely staining nuclei and was most intense in paired nuclei. CONCLUSION: The method of measuring Ki-67 expression by DAB staining combined with optical enhancement filters and quantification via computer-assisted image analysis techniques produced objective and reproducible results. As such, this method can offer (1) less intraobserver and interobserver variability, (2) a digital archival record, and (3) a baseline for digital exchange of information between studies.     

Analysis of DNA and morphometry in breast carcinoma

Feulgen-stained imprints and smears from 730 cases of invasive breast cancer were investigated using an image analysis system. From each tumor sample 100 cells were randomly scanned and several DNA and morphometrical parameters evaluated. Their prognostic value for a prediction of distant metastases within 5 years was investigated with the multivariate Cox regression analysis, which was performed for all consecutive cases, as well as for node-negative and node-positive patients separately. The multivariate analyses showed a strong prognostic value of the anisonucleosis (variation of nuclear radius) and the DNA histogram type in addition to the nodal status, the tumor size (pT), and the histological tumor grade. However, performing this analysis for both node-positive patients and for those without lymph node metastases demonstrated a different prognostic meaning of the variables. The combination of each of the group-specific variables led to a prognostic factor, which allowed an assignment of patients to several subgroups with significantly different risk for distant metastases. Thus, both a low-risk group of node-negative patients with a 5-year distant recurrence rate of only 5.8%, and a higher risk group of node-negative patients with a recurrence rate of 38.6% could be identified. Among the node-positive patients, a low-risk group with a distant recurrence rate of 8.6%, and also a high risk group with 69% distant recurrence, could be identified.     

Immunohistochemistry profiles of breast ductal carcinoma: factor analysis of digital image analysis data

Background

Molecular studies of breast cancer revealed biological heterogeneity of the disease and opened new perspectives for personalized therapy. While multiple gene expression-based systems have been developed, current clinical practice is largely based upon conventional clinical and pathologic criteria. This gap may be filled by development of combined multi-IHC indices to characterize biological and clinical behaviour of the tumours. Digital image analysis (DA) with multivariate statistics of the data opens new opportunities in this field.

Methods

Tissue microarrays of 109 patients with breast ductal carcinoma were stained for a set of 10 IHC markers (ER, PR, HER2, Ki67, AR, BCL2, HIF-1??, SATB1, p53, and p16). Aperio imaging platform with the Genie, Nuclear and Membrane algorithms were used for the DA. Factor analysis of the DA data was performed in the whole group and hormone receptor (HR) positive subgroup of the patients (n = 85).

Results

Major factor potentially reflecting aggressive disease behaviour (i-Grade) was extracted, characterized by opposite loadings of ER/PR/AR/BCL2 and Ki67/HIF-1??. The i-Grade factor scores revealed bimodal distribution and were strongly associated with higher Nottingham histological grade (G) and more aggressive intrinsic subtypes. In HR-positive tumours, the aggressiveness of the tumour was best defined by positive Ki67 and negative ER loadings. High Ki67/ER factor scores were strongly associated with the higher G and Luminal B types, but also were detected in a set of G1 and Luminal A cases, potentially indicating high risk patients in these categories. Inverse relation between HER2 and PR expression was found in the HR-positive tumours pointing at differential information conveyed by the ER and PR expression. SATB1 along with HIF-1?? reflected the second major factor of variation in our patients; in the HR-positive group they were inversely associated with the HR and BCL2 expression and represented the major factor of variation. Finally, we confirmed high expression levels of p16 in Triple-negative tumours.

Conclusion

Factor analysis of multiple IHC biomarkers measured by automated DA is an efficient exploratory tool clarifying complex interdependencies in the breast ductal carcinoma IHC profiles and informative value of single IHC markers. Integrated IHC indices may provide additional risk stratifications for the currently used grading systems and prove to be useful in clinical outcome studies.

Virtual Slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1512077125668949     

Effect of freezing on histologic grading of invasive ductal breast cancer

OBJECTIVE: To quantify the histologic changes caused by freezing during tissue processing and their influence on histologic malignancy grading as a prognostic factor in invasive ductal breast cancer. STUDY DESIGN: We studied frozen and nonfrozen formalin-fixed, paraffin-embedded samples of 18 cases of invasive ductal breast cancer. Features associated with histologic malignancy grading of breast cancer--i.e., nuclear pleomorphism, mitotic index and tubular differentiation--were assessed by quantitative morphometric methods. RESULTS: In our material, frozen samples consistently had a smaller mean nuclear profile area than nonfrozen samples (mean difference, 32%). Frozen nuclei were also clearly less symmetric and uniform in shape than non-frozen nuclei. Moreover, frozen samples had consistently higher mitotic indices than nonfrozen samples (mean difference, 66%, with the standardized mitotic index). Tubular differentiation, as expressed in fraction of fields with tubular differentiation, increased by 16% as a result of sample freezing. CONCLUSION: According to our results of morphometric measurement in invasive ductal breast cancer, great caution should be exercised when prognostic conclusions are based on frozen tissue samples.     

Expression of the serine protease, matriptase, in breast ductal carcinoma of Chinese women: correlation with clinicopathological parameters

Matriptase is a serine protease expressed by cells of surface epithelial origin, including epithelial breast tumor cells. Matriptase cleaves and activates proteins implicated in the progression of cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine matriptase expression in breast tumors of Chinese women and to identify its clinicopathological correlations. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 251 breast tumors including 30 fibroadenomas, 59 ductal carcinomas in situ (DCIS), 38 grade I invasive ductal carcinomas (IDC), 79 grade II IDC, and 45 grade III IDC. The matriptase scores were significantly higher in the tumors than their non-tumor counterparts (178+/-12 for fibroadenoma; 275+/-11 for DCIS; 299+/-10 for grade I IDC; 251+/-10 for grade II IDC; and 314+/-11 for grade III IDC). In cases of IDC, matriptase scores were significantly correlated with tumor staging and nodal staging. Our findings demonstrate that matriptase is over-expressed in breast ductal carcinoma of Chinese women. It therefore may be a good biomarker for diagnosis and treatment of malignant breast tumors.     

Fine needle aspiration cytology of glycogen-rich clear cell carcinoma of the breast: review of 37 cases with histologic correlation

OBJECTIVE: To analyze fine-needle aspiration cytology (FNAC) material from 37 cases of breast glycogen-rich clear cell cancer (GRCC) and correlate cytomorphologic features with histologic appearance to determine characteristics of GRCC on FNAC. STUDY DESIGN: We reviewed cytologic features of 37 cases of breast GRCC from the archives of Ege University Hospital diagnosed between 1994 and 2006. RESULTS: Thirty-seven patients with available aspirate and confirmed GRCC were identified. The female patients ranged from 32 to 81 years (mean 52 years). The initial cytologic diagnoses were adenocarcinoma for 27 and atypical or suspicious for cancer for 10. The cytologic picture was characterized by hypercellular tumor cells in loosely cohesive syncytial groups and some single cells. Most tumor cells had abundant, finely granular eosinophilic cytoplasm or foamy to clear cytoplasm with well-defined cytoplasmic membranes and moderate to marked nuclear pleomorphism with prominent nucleoli. Histologic examination confirmed all cases to be pure GRCC. ConCLUSION: Breast GRCC is a rare, distinct category with cytologic features that overlap considerably with those of other carcinomas. Awareness of variability in cytomorphologic appearance of GRCC and routine assessment for glycogen facilitate accurate diagnosis of these lesions by FNAC and enable prompt treatment of these poor-prognosis breast cancers.     

Epithelial myoepithelial carcinoma of the parotid gland with malignant ductal and myoepithelial components arising in a pleomorphic adenoma: a case report with cytologic, histologic and immunohistochemical correlation

BACKGROUND: To describe the cytologic, histologic and immunohistochemical findings of a case of epithelial myoepithelial carcinoma (EMC) arising from a pleomorphic adenoma (PA) of the parotid with both malignant epithelial and myoepithelial components. CASE: A 29-year-old female presented with a 1.5 x 1.5-cm, palpable mass of the left parotid of 7-8 months' duration with recent enlargement and pain. Fine needle aspiration biopsy (FNAB) revealed biphasic epithelial (small cell) and myoepithelial (large/clear cell) clusters arranged in a pseudopapillary and trabecular pattern with abundant hyaline material with many naked nuclei, together with areas typical of pleomorphic adenoma (PA) was noted. The cytology was reported as salivary gland neoplasm, "suggestive of adenoid cystic carcinoma, less likely pleomorphic adenoma." The mass was excised and histologically reported as "pleomorphic adenoma, with focal invasion of one resected margin." Four months later the tumor recurred, and FNAB showed almost the same cytologic features as did the previous aspirate. Due to early recurrence, previous histologic sections were reviewed, and typical areas of a biphasic pattern of EMC with atypicality and mitosis of both components was found. The final diagnosis was EMC ex PA. CONCLUSION: Although previous reports mention the difficulties in diagnosing EMC and differentiation from the more common salivary gland neoplasms such as PA, we like to emphasize the cytologic confusion that results when the tumors coexist.     

Maspin and c-erbB-2 expression in correlation with microvessel density in invasive ductal breast cancer

Maspin is a unique member of the serpin family involved in regulation of cell migration, apoptosis and angiogenesis in breast and prostate cancers. In this study maspin expression in comparison with c-erbB-2 (HER2/neu) oncogene expression and microvessel density was investigated. The examined material included specimens of primary invasive ductal breast cancer derived from 69 patients. They were analyzed immunocytochemically to assess maspin and c-erbB-2 expression, as well as microvessel density using endothelium marker CD31. In the studied cancers, maspin expression in cancer cells was detected in more than half of the cases (50.73%). Although statistically insignificant (p=0.27), maspin expression showed decreasing tendency with the increase of tumor grade. C-erbB-2 oncogene expression was observed in 78.26% of the examined cancers. Statistically significant positive correlation was found between c-erbB-2 expression and tumor grade (p<0.005). Analysis of the dependence between maspin and c-erbB-2 expression exhibited statistically significant inverse correlation (p<0.001). Mean microvessel density (MVD) of the studied cancers was 71.64 (SD=19.36). MVD decreased with the increase of maspin expression, whereas in the cases showing c-erbB-2 overexpression MVD was clearly higher. Both correlations were statistically significant (p<0.005). In conclusion, it could be stated that increase in maspin expression is associated with weaker expression of c-erbB-2 oncogene and lower microvessel density, which implies a significant role of maspin in tumor biology. However, the exact mechanism of maspin action (including its potential role in angiogenesis), as well as the assessment of its prognostic significance in breast cancer require further studies.     

Classifying breast carcinomas with DNA measurements and morphometry

The relation between the DNA distribution pattern and the variables DNA content and nuclear area was investigated in 73 breast carcinomas. According to the DNA patterns, the tumors were subclassified into groups I, II, III, and IV (Auer's classification). The Auer's classes correlate with prognosis. In this investigation it was shown that Auer's classes strongly correlate with the standard deviation of DNA content and mean nuclear area. The carcinomas were subclassified using data of DNA content and nuclear area. For the establishment of this subclassification, multivariate analysis was applied. It was evident that Auer's group III was a hybrid group. Therefore a second classification rule was calculated without the data of Auer's group III. This second classification rule was even more successful in determining the Auer's group of each individual case. Our investigation shows that it is possible to subclassify breast carcinomas objectively using DNA content and nuclear area of the tumor cells.     

Primary osteogenic sarcoma of the breast: cytomorphologic study of 3 cases with histologic correlation

BACKGROUND: Primary osteogenic sarcomas of the breast are extremely rare neoplasms. The histologic and cytologic features are comparable to those of their soft tissue and skeletal counterparts. To assess the utility of fine needle aspiration (FNA) in preoperative identification of osteogenic sarcomas, we retrospectively reviewed the FNA findings of 3 cases diagnosed in our hospital over 2 1/2 years. CASES: Three women, aged 48, 55 and 76 years, presented with a palpable lump of a few months' duration in their breasts. FNA was indicative of malignancy, and mastectomy with ipsilateral axillary lymph node dissection was performed. The cytologic features were of hypocellular or hypercellular smears with pleomorphic cells; scarce or abundant metachromatic amorphous material, suggestive of osteoid; osteoclast-like giant cells; and stromal fragments. CONCLUSION: Although cytologic features can be suggestive of osteosarcoma in the appropriate clinical setting, prompt preoperative diagnosis of malignancy in FNA samples of these tumors can avoid undertreatment, because mammographic and clinical findings are in many cases confused with the features of a benign lesion, more specifically calcified fibroadenoma.     

Recurrence following treatment of ductal carcinoma in situ with skin-sparing mastectomy and immediate breast reconstruction

Skin-sparing mastectomy with immediate breast reconstruction can provide an excellent cosmetic result. Despite its increasing popularity, few studies have assessed the risk of recurrence when the procedure is used for the treatment of ductal carcinoma in situ. To evaluate the oncologic safety of skin-sparing mastectomy used for the treatment of ductal carcinoma in situ, the recurrence rate was analyzed. Patients with ductal carcinoma in situ or invasive carcinoma or both who underwent skin-sparing mastectomy with immediate breast reconstruction between 1985 and 1994 and had a follow-up period of at least 6 years were included in this retrospective analysis. The recurrence rates were determined for invasive carcinoma (with or without foci of ductal carcinoma in situ) and ductal carcinoma in situ alone. A total of 221 patients were included, 177 patients with invasive carcinoma and 44 patients with ductal carcinoma in situ alone. The immediate breast reconstructions were performed with transverse rectus abdominis muscle (TRAM) flaps in 62 percent of patients, implants in 34 percent of patients, and latissimus dorsi myocutaneous flaps (with or without implants) in 4 percent of patients. The local recurrence rate was zero of 44 for patients with ductal carcinoma in situ and 5.6 percent (10 of 177) for patients with invasive carcinoma during a mean follow-up period of 9.8 years. There was a 6.8 percent (12 of 177) metastatic recurrence rate in the invasive carcinoma group. All recurrences were invasive ductal carcinoma. Of the patients with ductal carcinoma in situ alone, none developed metastatic disease. The combined metastatic and local recurrence rates for the invasive carcinoma group (n = 177) with each type of reconstruction were 13 percent (14 of 110), 12 percent (seven of 60), and 14 percent (one of seven) for TRAM flaps, implants, and latissimus dorsi flaps, respectively. The risk of recurrence following skin-sparing mastectomy and immediate breast reconstruction for ductal carcinoma in situ is low during this follow-up period. Therefore, skin-sparing mastectomy with immediate breast reconstruction seems to be a safe oncologic treatment option for ductal carcinoma in situ; however, a longer follow-up period is important to determine the long-term risk of recurrence.