Automated detection of cancer cells in effusion specimens by DNA karyometry.

BACKGROUND: The average sensitivity of conventional cytology for the identification of cancer cells in effusion specimens is only approximately 58%. DNA image cytometry (DNA-ICM), which exploits the DNA content of morphologically suspicious nuclei measured on digital images, has a sensitivity of up to 91% for the detection of cancer cells. However, when performed manually, to our knowledge to date, an expert needs approximately 60 minutes for the analysis of a single slide. METHODS: In the current study, the authors present a novel method of supervised machine learning for the automated identification of morphologically suspicious mesothelial and epithelial nuclei in Feulgen-stained effusion specimens. The authors compared this with manual DNA-ICM and a gold standard cytological diagnosis for 121 cases. Furthermore, the authors retrospectively analyzed whether the amount of morphometrically abnormal mesothelial or epithelial nuclei detected by the digital classifier could be used as an additional diagnostic marker. RESULTS: The presented semiautomated DNA karyometric solution identified more diagnostically relevant abnormal nuclei compared with manual DNA-ICM, which led to a higher sensitivity (76.4% vs 68.5%) at a specificity of 100%. The ratio between digitally abnormal and all mesothelial nuclei was found to identify cancer cell-positive slides at 100% sensitivity and 70% specificity. The time effort for an expert therefore is reduced to the verification of a few nuclei with exceeding DNA content, which to our knowledge can be accomplished within 5 minutes. CONCLUSIONS: The authors have created and validated a computer-assisted bimodal karyometric approach for which both nuclear morphology and DNA are quantified from a Feulgen-stained slide. DNA karyometry thus increases the diagnostic accuracy and reduces the workload of an expert when compared with manual DNA-ICM.

[Karyometry of BBN-induced precancerosis of the urothelium : An experimental analysis]. / Karyometrie BBN-induzierter Präkanzerosen des Urothels : Eine experimentelle Analyse.

BACKGROUND: The morphology of experimental precancerous lesions of the urinary bladder has been interpreted quite differently by various authors. OBJECTIVES: The aim of this investigation was to quantify these lesions by karyometry and, thus, to gain a more reliable understanding of the process. MATERIALS AND METHODS: A total of 60 Wistar rats were fed with N­butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at a concentration of 0.05 % in their drinking water to induce preneoplastic changes of the urothelium. After the second week of BBN exposition, 6 animals were killed every 2 weeks up to week 20. Smears of the scraped off urothelium of 3 urinary bladders of each group were analyzed cytologically and karyometrically. RESULTS: BBN exposition led to statistically significant changes of the karyometric values using the χ2 test to differentiate the control animals from the ones that had ingested BBN and the 2­week groups from each other. These changes consisted mainly in significant deviations of the size of the nuclear area within the different groups. CONCLUSION: Morphological and karyometrical analysis showed that biologically relevant stages in the development of chemically induced urothelial precancerous lesions could be realized much earlier than had been assumed in recent publications. Karyometric analysis offered a valid basis to describe the early morphologic alterations of carcinogenesis.

High Proportion of Nuclear Phenotype Identifies Aggressive Cutaneous Squamous Cell Carcinoma.

OBJECTIVE: To develop a quantitative histopathology algorithm to predict which patients with cutaneous squamous cell carcinoma (cSCC) were likely to experience recurrence or metastases. STUDY DESIGN: This retrospective study of cSCC lesions compared patients with aggressive disease (n = 40) and those with nonaggressive disease (n = 35). Based on a previous study using nuclear karyometry, we determined that aggressive lesions had a high proportion of a specific nuclear phenotype. The proportion of those nuclei was used to derive an aggressiveness score for each lesion. The mean age of patients was similar in both groups, as were the locations of index lesions. RESULTS: The mean aggressiveness scorefor cases with aggressive lesions was 0.60 ± 0.21 and was 0.28 ± 0.35 for those with nonaggressive lesions. The overall accuracy in properly characterizing lesions was 72%. The area under the receiver operating characteristic curve was 0.80 ± 0.05. In general, the aggressive nuclear phenotype is represented by elevated levels of chromatin clumps and short linear segments of dark chromatin/intense pixels. CONCLUSION: These data suggest that discriminant functions may be utilized to distinguish between aggressive and nonaggressive lesions at the time of diagnosis.

[Basic aspects of medical genetics]. / Grundlagen der medizinischen Genetik.

The human genome consists of 23 pairs of chromosomes that contain 20 000-25 000 genes. Genetic disorders can be caused by different mechanisms, and therefore the confirmation of a suspected diagnosis requires knowledge of the underlying defect, so that the correct test can be applied. Monogenic diseases are caused by disturbances in a single gene, and currently only targeted diagnostic testing is available following a specific clinical suspicion. Chromosomal disorders usually involve multiple genes, so that the symptoms are often less specific. Specialists in Medical Genetics FMH are trained in creating a clinical genetic differential diagnosis, requesting the according laboratory test, interpretating the results and providing expert genetic counseling in presymptomatic and prenatal diagnosis. In Switzerland, specific legal principles and ethical guidelines must be taken into account.

Le génome humain est constitué de 23 paires de chromosomes et contient 20 000 à 25 000 gènes. Les maladies génétiques peuvent être causées par différents mécanismes, or pour confirmer un diagnostic présumé et utiliser le test adéquat, il est nécessaire de connaître le défaut génétique sous-jacent. Les maladies monogéniques sont causées par des perturbations dans un seul gène, et actuellement seul un test diagnostique ciblé peut être réalisé suite à une suspicion clinique spécifique. Les anomalies chromosomiques impliquent généralement plusieurs gènes, donc les symptômes sont souvent moins spécifiques. Les spécialistes en génétique médicale FMH sont formés à reconnaître cliniquement les diagnostics génétiques, à demander les analyses correspondantes, à en interpréter les résultats et à donner un conseil génétique adapté en diagnostic présymptomatique et prénatal. En Suisse, des principes juridiques et éthiques spécifiques doivent également être pris en compte.

Karyometric comparison of splenic and gastric marginal zone lymphomas.

BACKGROUND: Marginal zone lymphomas are indolent B-cell lymphomas associated with autoimmunity and chronic inflammation. The two most frequent variants are mucosa associated lymphoid tissues marginal zone lymphomas and splenic marginal zone lymphomas. The aim of the study was to determine if it is possible to classify splenic and gastric lymphomas according to karyometric features. METHODS: The material consisted of 16 splenic and 14 gastric lymphomas. The measurements were done with the AnalySIS image analysis system. In each case at least 100 nuclei were selected, and 19 different geometric parameters were measured. RESULTS: On statistical analysis, the nuclei of splenic and gastric lymphomas showed differences in most parameters, but significant overlap of the values was present. Neural networks were trained and used for classification of the data. By this method, the nuclei were properly classified with a sensitivity of 0.75 and specificity of 0.71. In addition, in all the cases the majority of the nuclei were properly classified, thus allowing correct classification of all the cases into "splenic" or "gastric". CONCLUSION: These results support the view that mucosa-associated lymphoid tissue lymphomas and splenic marginal-zone lymphomas are separate entities.

Differential diagnosis of follicular tumor by expert systems based on a set of quantitative features of thyrocyte nuclei and aggregates.

OBJECTIVE: To develop expert systems for classification of follicular thyroid tumor at a preoperative stage. STUDY DESIGN: Fine needle aspiration biopsy of the thyroid gland with a histologic conclusion of follicular cancer and follicular adenoma were the object of the morphometric study. General sample size was 4500 nuclei and 3000 aggregates. RESULTS: Quantitative regularities of pathologic changes in thyrocyte nuclei and aggregates in follicular cancer and follicular adenoma were revealed. Threshold values and weighting coefficients of quantitative features of thyrocyte nuclei and aggregates characterizing cancer made the basis of the two expert systems. Expert systems included standard 2-D S-matrix containing threshold values of nuclei and aggregates in cancer and their weighting coefficients as well as 1-D scientific X-matrix designed for filling with quantitative features of the studied object. The diagnosis was verified by the value of a diagnostic index by means of comparing feature values in the corresponding elements of S- and X-matrices. After that, a diagnostic index was calculated taking into account the features' weighting coefficient. CONCLUSION: The developed expert systems based on a set of quantitative features of thyrocyte nuclei and aggregates will allow assessing the malignant potential of a follicular thyroid tumor at a preoperative stage.

Automation of the buccal micronucleus cytome assay using laser scanning cytometry.

Laser scanning cytometry (LSC) can be used to quantify the fluorescence intensity or laser light loss (absorbance) of localized molecular targets within nuclear and cytoplasmic structures of cells while maintaining the morphological features of the examined tissue. It was aimed to develop an automated LSC protocol to study cellular and nuclear anomalies and DNA damage events in human buccal mucosal cells. Since the buccal micronucleus cytome assay has been used to measure biomarkers of DNA damage (micronuclei and/or nuclear buds), cytokinesis defects (binucleated cells), proliferative potential (basal cell frequency), and/or cell death (condensed chromatin, karyorrhexis, and pyknotic and karyolytic cells), the following automated LSC protocol describes scoring criteria for these same parameters using an automated imaging LSC. In this automated LSC assay, cells derived from the buccal mucosa were harvested from the inside of patient's mouths using a small-headed toothbrush. The cells were washed to remove any debris and/or bacteria, and a single-cell suspension prepared and applied to a microscope slide using a cytocentrifuge. Cells were fixed and stained with Feulgen and Light Green stain allowing both chromatic and fluorescent analysis to be undertaken simultaneously with the use of an LSC.

Nuclear grading of primary pulmonary adenocarcinomas: correlation between nuclear size and prognosis.

BACKGROUND: According to the World Health Organization Classification of Tumors, the prognostic value of morphometric cytologic atypia has not been assessed in pulmonary adenocarcinoma. METHODS: Primary tumors of 133 pulmonary adenocarcinomas

Pivotal karyometric measurements in different types of cardiomyopathic morphology: study of hearts explanted from transplant recipients.

BACKGROUND: Morphometric studies based on the measurement of cardiocyte nuclei have focused on progressive hypertrophy rather than shape, which is a deciding factor for the diagnosis of hypertrophy in myocardial diseases. The aim of this research was to demonstrate how the digital morphology of cardiocyte nuclei change correlated with the type of myocardial pathology. MATERIALS AND METHODS: The study groups encompassed 7 hearts with dilated cardiomyopathy (DCM) and 8 hearts with ischemic heart disease (IHD) which were explanted. A comparative group consisting of myocardial hypertrophy was contrasted with a control group of donor heart fragments. Cardiocyte nuclei were evaluated morphometrically on histologic slides. We calculated the nuclear area, length, breadth, perimeter, roundness, elongation, fullness factors, and nuclear chromatin mean gray level. The results were subjected to discriminant analysis. RESULTS: All karyometric measurements analyzed by backward discriminant analysis showed only 2 powerful factors: nuclear breadth and chromatin mean gray level. The Mahalanobis distance showed the proximity of control and hypertrophy groups, whereas differences between IHD and DCM were nonsignificant. CONCLUSION: The lack of karyometric differences between IHD and DCM suggested a common morphologic response for long-lasting progressive injury. The main morphologic differences were dependent on nuclear chromatin activity/stainability and nuclear breadth, suggesting darker and thinned nuclei in normal and adaptative stages and irregular brighter nuclei in cardiomyopathies.

Karyometry: correction algorithm for differences in staining.

OBJECTIVE: To describe an algorithm that allows the correction of differences in staining of histopathologic sections while preserving chromatin texture. STUDY DESIGN: In order to preserve the texture of the nuclear chromatin in the corrected digital imagery, it is necessary to correct the images pixel for pixel. This is accomplished by mapping each pixel's value onto the cumulative frequency distribution of the data set to which the image belongs, to transfer to the cumulative frequency distribution of the data set serving as standard and to project the intersection down onto the pixel optical density scale for the corrected value. RESULTS: Feature values in the corrected imagery, for the majority of features used in karyometry, are between < 1% and a few percent of the feature values in standard imagery. For some higher-order statistical features involving multiple pixels, sensitivity to a shift in the cumulative frequency distribution may exist, and a secondary small correction by a factor may be required. CONCLUSION: The correction algorithm allows the elimination of the effects of small staining differences on karyometric analysis.

Automatic segmentation of cell nuclei in bladder and skin tissue for karyometric analysis.

OBJECTIVE: To automatically segment cell nuclei in histology images of bladder and skin tissue for karyometric analysis. STUDY DESIGN: The 4 main steps in the program were as follows: median filtering and thresholding, segmentation, categorizing and cusp correction. This robust segmentation technique used properties of the image histogram to optimally select a threshold and create closed 4-way chain code nuclear segmentations. Each cell nucleus segmentation was treated as an individual object of which the properties of segmentation quality were used for criteria to classify each nucleus as: throw away, salvageable or good. An erosion/dilation procedure and rethresholding were performed on salvageable nuclei to correct cusps. RESULTS: Ten bladder histology images were segmented both by hand and using this automatic segmentation algorithm. The automatic segmentation resulted in a sensitivity of 76.4%, defined as the percentage of hand-segmented nuclei that were automatically segmented with good quality. The median proportional difference between hand and automatic segmentations over 42 nuclei each with 95 features used in karyometric analysis was 1.6%. The same procedure was performed on 10 skin histology images with a sensitivity of 83.0% and median proportional difference of 2.6%. CONCLUSION: The close agreement in karyometric features with hand segmentation shows that automated segmentation can be used for analysis of bladder and skin histology images.

Progression of skin lesions from normal skin to squamous cell carcinoma.

OBJECTIVE: To assess the changes in the nuclear chromatin pattern concomitant with progressive sun damage in skin biopsies ranging from sun-exposed, normal-appearing skin to squamous cell carcinoma (SCC). STUDY DESIGN: Biopsies were taken from 140 cases with sun-exposed but histopathologically normal skin, from 20 cases visually assessed as pre-actinic keratosis (pre-AK) or early AK, from 30 cases of AK, and from 21 cases of SCC. A total of 21,094 nuclei were recorded from these biopsies. High-resolution digital imagery was recorded, and features descriptive of the nuclear chromatin pattern were computed. Both supervised learning and unsupervised learning algorithms were employed to derive progression plots. RESULTS: With increased sun exposure, the proportion of nuclei exhibiting changes in the nuclear chromatin pattern rises notably. Using karyometry, no significant differences could be substantiated between nuclei collected from early AK sites and AK lesions. Cases of SCC fell into 2 distinct groups. A larger group (approximately 66.7% of cases) had characteristics similar to AK. A smaller group (approximately 33.3% of cases) represented much more progressed lesions. CONCLUSION: Karyometric assessment can provide a numeric measure of progression for sun damage and of the deviation from normal in both AK and SCC lesions.

Estudio cariométrico de placentas de ratones con infección aguda por diferentes cepas de Trypanosoma cruzi / Karyometric study of placentas from mice with acute infection by different strains of Trypanosoma cruzi

El objetivo de este trabajo fue evaluar cariometricamente las alteraciones causadas por diferentes cepas de T. cruzi en la placenta del ratón. Ratones hembras de 60 días, grávidas, fueron inoculadas, intraperitonealmente, con 2 x 10(5) tripomastigotes sanguíneos de las cepas colombiana, Y, Solivia o RC del T. cruzi. Fueron observadas claras diferencias en las alteraciones cariométricas de las células trofoblásticas gigantes y de las células trofoblásticas de la zona esponjosa. Los resultados demostraron que las cepas colombiana y RC causan alteraciones tanto en las células trofoblásticas gigantes como en las células del trofoblasto esponjoso, mientras que las cepas Y y Bolivia provocan alteraciones solamente en las células trofoblásticas gigantes. Es posible concluir que cada cepa posee características propias y que, a pesar del tipo similar de transmisión, presenta matices diferenciales en el proceso de la patogénesis placentaria.

The objective of this work was to evaluate karyometrically the alterations caused by different strains of Trypanosoma cruzi in the mouse placenta. Pregnant mice, 60-day old, were intraperitoneally inoculated with 2 x 10(5) bloodstream trypomastigotes of the Colombian, Y, Bolivia or RC strain of T cruzi. There were observed clear differences in the karyometric alterations of the trophoblast giant cells and in the spongiotrophoblast cells. The results demonstrate that the Colombian and RC strains cause alterations both in the trophoblast giant cells and in the spongiotrophoblast cells, whereas the Y and Bolivia strains provoke alterations only in the trophoblast giant cells. It is possible concluding that each strain has its own characteristics and that, in spite of the similar type of transmission, it show differential nuances in the placental pathogenic process.

Limits of detection of chemopreventive efficacy: karyometry of skin biopsies.

OBJECTIVE: This study was designed to establish estimates of the smallest effects due to chemopreventive intervention detectable by karyometry in skin biopsies. METHODS: Estimates of the smallest change of statistical significance and estimates of the power of the test were derived for several key features descriptive of the distribution of nuclear chromatin. Results from triplicate biopsies from the same case were used to provide estimates of the within-case, biopsy-to-biopsy variance. RESULTS: Generally, a change in feature value due to chemopreventive intervention can be statistically secured when it amounts to 5% to 10%. In clinical trials where matched baseline and end of study biopsies from the same cases are available, paired comparison ANOVA can detect a 2% change on samples of 25 cases. Establishing efficacy in individual cases requires a change in feature values on the order of 10% to 15%. CONCLUSIONS: Karyometry provides a sensitive, quantitative method for the assessment of efficacy of chemoprevention. The effects of within-case, biopsy-to-biopsy variance need to be considered only in the evaluation of individual cases and are on the order of 5% in skin biopsies.

Potential role of nuclear appearance in pathologic recognition and delimitation of sessile serrated polyps of the colon: a karyometric study.

OBJECTIVE: To estimate the extent to which surgical pathologists can rely on abnormal nuclear appearance to recognize sessile serrated adenoma (SSP) and to define its extent. STUDY DESIGN: Digitized images of nuclei of superficial crypt cells from SSPs, banal hyperplastic polyps (BHPs), tubular adenomas (TAs) and normal colonic mucosa (N) in surgical pathology specimens were analyzed for size, shape, area, optical density (summed and average) and 22 Markovian texture characteristics. RESULTS: Statistical classification functions correctly distinguished TA from N nuclear image profiles in 93.3% of cases, SSP from N in 70.0% and BHP from N in 74.1%. SSP nuclear feature vectors were less effectively separated from N than BHP and TA on discriminant analysis of the combined data set, and correct classification was achieved in 79.6% of TAs, 53.5% of SSPs and N and 64.2% of BHPs. CONCLUSION: Karyometry distinguished stained nuclei of TAs, but had limited ability to separate nuclei of SSP from those of N and BHP. This suggests that the pathologist attempting to diagnose or delimit the margins of an SSP will find nuclear appearances less helpful than when examining a TA.

Karyometry of breast epithelial cells acquired by random periareolar fine needle aspiration in women at high risk for breast cancer.

OBJECTIVE: To establish whether karyometry was likely to detect change in the proportion of abnormal cells in random periareolar fine needle aspiration (RPFNA) specimens from high-risk women in a 6-month prevention trial with an aromatase inhibitor. STUDY DESIGN: Papanicolaou-stained ThinPrep slides of RPFNA samples from 11 of 42 women were digitally recorded at high resolution, with 200 cells measured per slide, at baseline (BL) and at the end of study (ES) after 6 months. The nuclear chromatin pattern characteristics were assessed by multivariate analytic techniques; determination of nuclear abnormalities was performed and cells that showed expression of abnormality were identified. RESULTS: The BL FNA samples contain approximately 90% cells with a chromatin pattern as expected in a normal cell population. A small subpopulation of cells had deviations from normal. At ES the proportion of these cells was reduced, to a statistically significant degree,from < 10% to 2-5%. CONCLUSION: Nuclear karyometry is a promising technique for characterizing the proportion of cells deviating from normal in cytologic specimens and should be explored further as an intermediate endpoint in prevention trials.

Chromatin phenotype karyometry can predict recurrence in papillary urothelial neoplasms of low malignant potential.

BACKGROUND: A preceding exploratory study (J. Clin. Pathol. 57(2004), 1201-1207) had shown that a karyometric assessment of nuclei from papillary urothelial neoplasms of low malignant potential (PUNLMP) revealed subtle differences in phenotype which correlated with recurrence of disease. AIM OF THE STUDY: To validate the results from the exploratory study on a larger sample size. MATERIALS: 93 karyometric features were analyzed on haematoxylin and eosin-stained sections from 85 cases of PUNLMP. 45 cases were from patients who had a solitary PUNLMP lesion and were disease-free during a follow-up period of at least 8 years. The other 40 were from patients with a unifocal PUNLMP, with one or more recurrences in the follow-up. A combination of the previously defined classification functions together with a new P-index derived classification method was used in an attempt to classify cases and identify a biomarker of recurrence in PUNLMP lesions. RESULTS: Validation was pursued by a number of separate approaches. First, the exact procedure from the exploratory study was applied to the large validation set. Second, since the discriminant function 2 of the exploratory study had been based on a small sample size, a new discriminant function was derived. The case classification showed a correct classification of 61% for non-recurrent and 74% for recurrent cases, respectively. Greater success was obtained by applying unsupervised learning technologies to take advantage of phenotypical composition (correct classification of 92%). This approach was validated by dividing the data into training and test sets with 2/3 of the cases assigned to the training sets, and 1/3 to the test sets, on a rotating basis, and validation of the classification rate was thus tested on three separate data sets by a leave-k-out process. The average correct classification was 92.8% (training set) and 84.6% (test set). CONCLUSIONS: Our validation study detected subvisual differences in chromatin organization state between non-recurrent and recurrent PUNLMP, thus allowing a very stable method of predicting recurrence of papillary urothelial neoplasms of low malignant potential by karyometry.

Accelerated nuclei preparation and methods for analysis of histone modifications in yeast.

The continuing identification of new histone post-translational modifications and ongoing discovery of their roles in nuclear processes has increased the demand for quick, efficient, and precise methods for their analysis. In the budding yeast Saccharomyces cerevisiae, a variety of methods exist for the characterization of histone modifications on a global scale. However, a wide gap in preparation time and histone purity exists between the most widely used extraction methods, which include a simple whole cell extraction (WCE) and an intensive histone extraction. In this work we evaluate various published WCE buffers for their relative effectiveness in the detection of histone modifications by Western blot analysis. We also present a precise, yet time-efficient method for the detection of subtle changes in histone modification levels. Lastly, we present a protocol for the rapid small-scale purification of nuclei that improves the performance of antibodies that do not work efficiently in WCE. These new methods are ideal for the analysis of histone modifications and could be applied to the analysis and improved detection of other nuclear proteins.

Analysis of karyometric variables in spontaneously occurring canine mammary tumors.

OBJECTIVE: To study alteration in karyometric variables to distinguish benign from malignant canine mammary tumors. STUDY DESIGN: Formalin-fixed, paraffin-embedded tissue sections from 16 cases of canine mammary tumors were stained with hematoxylin-eosin and analyzed for mean nuclear area, mean nuclear perimeter and shape factor. The morphometric descriptors were then correlated with conventional histologic diagnosis. RESULTS: On Mann-Whitney U-test, nuclear variables, that is, mean nuclear area and mean nuclear perimeter, revealed significant difference between malignant and benign groups (p < 0.01), while shape factor showed significant difference at p < 0.05. All morphometric descriptors showed significant correlation with each other and with histopathologic diagnosis. Contrary to expectations, shape factor was lower in benign compared to malignant tumors. In benign mammary tumors, the highest mean nuclear area and perimeter were 39.59 +/- 10.34 microm2 and 25.39 +/- 4.14 microm, respectively, for fibroadenoma; and in malignant tumors the highest mean nuclear area and perimeter were 48.44 +/- 12.89 microm2 and 27.36 +/- 4.31 microm, respectively, for papillary adenocarcinoma. CONCLUSION: This study provides insight into the alterations in karyometric variables in canine mammary tumors. Mean nuclear area and perimeter appear to have potential to differentiate benign and malignant canine mammary tumors.

Karyometry of infiltrating breast lesions.

OBJECTIVE: To characterize nuclei from well-differentiated, moderately differentiated and poorly differentiated lesions of invasive breast cancer by karyometry and to test the hypothesis that these diagnostic categories form homogeneous sets. STUDY DESIGN: Histopathologic sections from 6 cases of well-differentiated, 11 cases of moderately differentiated and 17 cases of poorly differentiated ductal carcinomas were digitally recorded. From each case 100 nuclei were segmented and analyzed by karyometry. A discriminant analysis was performed, and nuclear and lesion signatures were computed. The nonsupervised learning algorithm P-index was applied. A progression curve per diagnostic category based on mean nuclear abnormality and a discriminant function score was derived. RESULTS: The well-differentiated lesions formed a homogeneous set, but both the moderately and poorly differentiated lesions showed 2 significantly different subpopulations with nuclei of substantially different nuclear abnormality and progression. CONCLUSION: The visual histopathologic diagnostic assessment of these lesions was based on an evaluation of both tissue architectural criteria and nuclear criteria. Here, only the pattern of nuclear chromatin was evaluated. Cases belonging to the same diagnostic category as assessed by their differentiation may be further characterized by the extent to which the nuclei deviate from normal. There was substantial case-to-case heterogeneity in these invasive lesions.