Pure ductal carcinoma in situ and in situ component of ductal invasive carcinoma of the breast. A preliminary morphometric study.

Nuclear pleomorphism is a fundamental feature in evaluating the aggressiveness of ductal carcinoma in situ (DCIS) of the breast. In this study, pure DCIS and the in situ component (IS-comp) of invasive duct carcinoma (IDC) are compared in order to verify if these are two different entities or the same process observed at different times during its evolution. Five cases of pure DCIS and nine of IDC with extensive in situ component were selected. They were moderately and poorly differentiated. 30 nuclei for each DCIS, and 30 nuclei for both the in situ and invasive component of each IDC were studied; thus, a total of 720 nuclei were submitted to the SAM (Shape Analytical Morphometry) analysis, which enables a numerical expression not only of dimensions (area, perimeter, diameter) but also of nuclear contour irregularities and nuclear shape distortions. Univariate statistical comparisons were carried out between the nuclei of: (1) DCIS and in situ component of invasive duct carcinoma, (2) DCIS and the invasive component of infiltrating carcinoma and (3) between the in situ and invasive component of infiltrating carcinoma. Multivariate analysis was utilized to compare nuclei of DCIS with the in situ component of IDC. The in situ features of each tumor were also evaluated with the mitotic index (MI). Nuclei of pure DCIS resulted significantly larger (p < 0.001) and with a more regular shape (p < 0.001) than those of the in situ component of IDC. No differences were observed between the nuclei of the in situ and the invasive component of infiltrating carcinomas. Multivariate statistical analysis discriminated 77% of nuclei of in situ proliferation when both G2 and G3 tumors were considered, and 80% when only G3 tumors were considered. In conclusions morphological differences exist between pure DCIS and the in situ component of IDC, which may be an expression of their biological behavior; moreover, these morphological differences seem to have a better discriminating power within the same histological grade.

Quantitative morphometrical investigation of basal cell layer in laryngeal premalignant lesions.

Histological diagnosis of laryngeal dysplasia is quite subjective. Since morphometry is highly reproducible, this method was applied to compare shape and size variations of the basal nuclei of the laryngeal epithelium in normal, laryngeal intraepithelium neoplasia (LIN) and invasive carcinoma to assess the reliability of light microscopic criteria used in grading dysplasia according to Friedman classification. Morphometrical analysis was carried out by Shape Analytical Morphometry (S.A.M) system. The logical architecture assumes that each irregular shape contains elements of two distinct logical domains: gross distortions that interest the contour and its local perturbations. These features were investigated separately by analytical procedures to acquire independent parameters both on the logical level and the numerical one. The nuclear area significantly increased from normal to carcinoma (p<.001). The increasing of the nuclear area was evident also in LIN I. Nuclear distortions were present in LIN II and LIN III. The highest nuclear contour irregularities were found in LIN III. Multivariate analysis showed a difficulty in discriminating various grades of dysplasia, especially between LIN I and LIN II (31% of error). In conclusion, our results indicate that nuclear pleomorphism of the basal cells layer, using a unique evaluator, is an unsatisfying criterion to distinguish moderate dysplasia.

[The Ljubljana classification of epithelial hyperplastic laryngeal lesions: a morphometric evaluation of nuclear pleomorphism]. / La classificazione di Ljubljana delle lesioni iperplastiche dell'epitelio laringeo: valutazione morfometrica del pleomorfismo nucleare.

Epithelial hyperplastic laryngeal lesions (EHLL) are associated, with a varying degree of "epithelial risk"- to develop invasive carcinoma. Several classifications have been proposed but none has received a total agreement. The 1999 Ljubljana classification distinguished four grades: simple, abnormal and atypical hyperplasia and in situ carcinoma (ISC). The first two grades are considered benign lesions; the ISC is the malignant lesion, while the atypical hyperplasia is considered a "risky lesion". This is characterized by alterations of epithelial cells towards malignancy, but not to the extent to be found in carcinoma cells. Such characteristics refer to cytomorphological (e.g., nuclear hyperchromatism, nucleoli, increased nuclear/cytoplasmic ratio) and architectural (e.g. stratification, orientation, maturation) features. In the Ljubljana scheme, nuclear pleomorphism is one of the most important features. We wanted to improve the importance of nuclear pleomorphism in the basal cells layer in different classes of EHLL using morphometrical analysis. We studied 8 cases of simple hyperplasia, 10 of abnormal hyperplasia, 10 of atypical hyperplasia and 8 of ISC using the software SAM (Shape Analytical Morphometry). The results were submitted to univariate statistical analysis. Nuclear dimensions (maximum diameter, perimeter and area) showed a progressive increase from simple to atypical hyperplasias to ISC, while abnormal hyperplasia showed the lowest values. On the contrary, analytical parameters related to nuclear contour irregularities and asymmetries showed their highest values in abnormal hyperplasia nuclei. There were no significant differences between atypical hyperplasia and ISC, while it was possible to differentiate abnormal hyperplasia from the others. In conclusion basal nuclei of atypical hyperplasia and ISC are similar so that other cytological and morphological architectural parameters are necessary to distinguish the two lesions. Abnormal hyperplasia seems to be the biological category of 'proliferative " benign laryngeal epithelium; simple hyperplasia refers to "stable" - irritative epithelium.

Morphometric discrimination between syndromic (Gorlin) and nonsyndromic keratocysts.

OBJECTIVE: To search for morphologic nuclear features in the epithelial lining of odontogenic keratocysts to differentiate simple from Gorlin syndrome cysts. STUDY DESIGN: Five cases of syndrome-associated keratocysts and five of simple ones were studied. Thirty nuclei from the epithelial basal layer for each case were analyzed by the shape analytical morphometry (SAM) software system to quantitatively evaluate nuclear dimensions (area, perimeter, diameter), contour irregularities and nuclear shape asymmetries. Results were subjected to Student's t test and cluster analysis. RESULTS: Values of nuclear dimensions were very close in both groups of keratocysts, without any significant statistical differences. The variables related to nuclear profile irregularities, as well as those describing nuclear asymmetry, showed significantly higher values (P < .001) in syndromic cysts. Cluster analysis produced two different clusters by using variables related to nuclear contour irregularities. CONCLUSION: Preliminary results indicate the existence of nuclear morphologic differences between simple and syndromic cysts.

Real-time quantification of the proliferative state in astrocytomas.

OBJECTIVE: To evaluate proliferative activity in a set of gliomas and to compare the quantitative data obtained by a real-time processor with the labelling index (LI) and mitotic index (MI). STUDY DESIGN: Ki-67 immunostaining was performed on paraffin-embedded specimens from 42 cases of glioblastomas, 17 cases of anaplastic astrocytomas and 14 cases of low grade astrocytomas. Nuclear positivity was calculated as LI and by a real-time image processor for quantitative evaluation. MI was also calculated at 10 high-power fields. The data obtained from glioblastomas were compared with those from anaplastic and low grade astrocytomas. To all the data was applied the Pearson test to verify the correlation between counting and quantitative values and between proliferative markers and survival. RESULTS: A positive trend from low grade astrocytomas to glioblastomas was found for Ki-67 (LI and quantitative values) and MI, with highly significant differences between the three grades of gliomas considered. A good correlation between LI and quantitative values of Ki-67 was found. Very little relationship resulted between survival and Ki-67 LI. No relationship was found between survival and quantitative values of Ki-67. CONCLUSION: Ki-67 allowed effective separation of astrocytic tumors with different grades of malignancy. Quantitative evaluation of color information by means of a real-time processor proved to be a useful, objective and fast way to obtain readings, useful for grading purposes but not for prognostic evaluation.

[Atypical meningioma: morphometric analysis of nuclear pleomorphism]. / Meningioma Atipico: un'analisi morfometrica del polimorfismo nucleare.

The revised edition of the WHO classification of brain tumours now includes the "atypical" meningioma (grade II) which should be placed between the common type (grade I) and anaplastic type (grade III) according to histomorphological features and prognosis. However, diagnostic criteria for atypical meningioma are vague and the significance of brain invasion in the determination of malignancy is controversial. Nuclear pleomorphism and mitoses are usually considered the most important parameters to distinguish atypical and malignant meningiomas. According to WHO classification we selected eight cases of meningioma diagnosed as atypical (3 cases) and malignant (5 cases). All the tumours were supratentorially located. Nine cases of benign meningiomas were also studied as a control group. Morphometrical analysis was carried out by S.A.M. (Shape Analytical Morphometry) system. S.A.M. logical architecture assumes that each irregular shape contains elements of two distinct logical domains: gross distortions that interest the contour and its local perturbations. These features were investigated separately by analytical procedures to acquire independent parameters both on the logical and the numerical level. The results, statistically evaluated, show that nuclear pleomorphism is not a satisfactory criterion, if used alone, to distinguish atypical from malignant meningioma (Discriminant Analysis: 19% of minimum error).

Somatostatin expression in TS16 mouse brain cultures.

Somatostatin expression in trisomy 16 mouse neuronal cultures has been studied to investigate the effects of the presence of an extra copy of the pre-pro-somatostatin (ppSS) gene on mouse chromosome 16. The immunoreactivity for somatostatin (SS) was considered in mixed cultures of neurons and glia cells and in neuron-enriched cultures as well as that for neuropeptide Y, glutamic acid decarboxylase, and gamma-enolase immunoreactivity the genes of which are not present on mouse chromosome 16. ppSS and pre-pro-neuropeptide Y (ppNPY) mRNA expression was evaluated and SS immunoreactivity in neurons analyzed by a morphometrical study. The extra copy of the ppSS gene resulted in a significantly increased level of the transcript in trisomic cultures, whereas the expression of the other neuropeptides did not differ. The absence of glial cells in these cultures reduced the number of SS-positive neurons making their number comparable in the trisomic and control cultures. Thus, in spite of higher expression of the ppSS mRNA in trisomic cultures, the determination of this peptidergic phenotype was influenced by the presence of neuroglial cells.

[Malignant mesothelioma of the pleura. Morphometric study]. / Il mesotelioma maligno della pleura. Studio morfometrico.

Malignant mesothelioma is difficult to distinguish from other pleural malignancies and also from benign mesothelial lesions. A morphometric study has been performed to distinguish between them using quantitative size and shape parameters. Seven cases of malignant mesothelioma, 5 cases of pleural metastatic adenocarcinoma and 4 cases of benign mesothelial lesions were selected and subjected to S.A.M. (Shape Analytical Morphometry). The results, statistically evaluated, showed that morphometric parameters can be proposed for diagnostic purposes being useful in the discrimination among the three populations. In fact, multivariate discriminant analysis (MDA) of the quantitative parameters obtained by morphometrical study distinguished the three groups of lesions with only 2% of error between BML/MM, 7% of error between BML/MA and 25% between MM/MA.

Nuclear morphometry in node-negative breast carcinoma.

OBJECTIVE: To determine whether nuclear morphometry can confirm or add useful information to classic clinicopathological prognosticators to identify the subpopulation of breast carcinoma patients with node-negative (N-) disease, at high risk of disease relapse. STUDY DESIGN: On the basis of results obtained by clinicopathologic evaluation of a group of patients with N- breast cancer, on a test group of 56 cases (32 patients disease free and 24 with relapse), we performed a morphometric analytical study of nuclei using the Shape Analytical Morphometry (SAM) software system; 20 nuclei for each case and 17 morphometric parameters for each nucleus were analyzed. RESULTS: The SAM system allowed us to quantify shape differences in nuclei in terms of contour irregularities and asymmetries along with evaluation of nuclear dimensions. Dimensional and analytic parameters were subjected to univariate (Student's t test) and multivariate (Hotelling's test) analysis. Multivariate discriminant analysis showed that an exact forecast of disease relapse could be made in 77% of patients with N- breast cancer by using a set of six both analytic and dimensional parameters. CONCLUSION: These results confirm that nuclear pleomorphism is the result of both contour irregularities and shape asymmetries and that even though they should be considered preliminary results, they stress the importance of quantitative shape evaluation.

Recurring intracranial meningiomas. Morphometrical evaluation of nuclear pleomorphism by S.A.M. (shape analytical morphometry) system.

Meningiomas are the most common neoplasms of the central nervous system and their biological behavior is not always predictable from the histologic appearance of the tumors. The nuclear pleomorphism seems to be one of the most important morphological features in the prediction of recurrence. By using analytical morphometric methods it is possible to quantify nuclear atypias and to obtain parameters describing nuclear contour irregularities and distortions of the figure. Moreover the amount of information obtained from analytical procedure allowed to discriminate, by multivariate discriminant analysis recurrent or no-recurrent meningiomas (5% of error).

Nuclear morphometry in squamous cell carcinoma (SCC) of the tongue.

In the histological grading of oral squamous cell carcinoma (OSCC) nuclear features are very important. Nevertheless evaluation is usually performed in a subjective and not highly reproducible way. The aim of this work was to investigate the relationship between nuclear shape and survival in 30 cases of carcinoma of the tongue. All the patients were divided into two groups: short-term survival and long-term survival. Twenty nuclei for each tumour were submitted to a morphometrical study by the shape analytical morphometry (SAM) software system. It was thus possible to evaluate not only nuclear dimensions but also nuclear contour irregularities and nuclear shape asymmetries. Multivariate discriminant analysis (MDA) of the quantitative parameters obtained by the morphometrical study distinguished the patients of the two groups with only a 10% error; moreover successful cluster analysis was performed by using Fourier parameters. Both these sets of results were achieved mainly owing to the parameters for contour irregularities.

Size and shape evaluation of astrocytoma nuclei with the shape analytical morphometry software system.

Eighteen cases of astrocytomas diagnosed as grades 1-3 of malignancy were studied with analytical morphometry to determine if different grades can be discriminated by size and shape parameters related to the nuclei. The shape analytical morphometry system was used to calculate dimensional measurements and express shape irregularities through quantitative parameters. With the shape asymmetry evaluator procedure, nuclear symmetry was evaluated. All parameters subjected to univariate and multivariate strategies were the same in easily distinguishing grade 1 from the others. Grades 2 and 3 could not be discriminated completely by either dimensional or with analytical parameters revealing strong similarities of nuclear shape and dimensions.

Nuclear morphology and histological grading of oral squamous cell carcinoma (OSCC). A morphometric study.

Histological grading of oral squamous cell carcinoma (OSCC) is usually evaluated in a semiquantitative way and this causes a low grade of reproducibility of this prognostic evaluation. The aim in this preliminary phase is to investigate the relation between nuclear shape (studied by an analytical procedure) and histological grading to evaluate the weight of nuclear shape and dimension in grading formulation. Nine cases of OSCC were studied (3 cases of G1, G2 and G3 respectively) with 20 nuclei for each case. All the tumors were localized on the lip. Histologic grading according to the Bryne system was performed. Each nucleus was submitted to a morphometrical study by the SAM (Shape Analytical Morphometry) system which, apart from dimensions allows us to evaluate nuclear contour irregularities and nuclear shape asymmetry. Nuclei of squamous cell carcinoma show a progressive increase in dimensions (area, perimeter and diameter) from G1 to G3. Nuclei of G3 carcinoma are characterized by the most irregular shape. Size differences (mean value) are significant to differentiate G1 nuclei from G2 and G3 nuclei while shape parameters are useful in distinguishing G3 nuclei from G1 and G2. Multivariate discriminant analysis showed that by using only nuclear parameters it is possible to correctly classify about 80% of nuclei. This study, using quantitative methods, confirmed nuclear pleomorphism to be the most important feature in grading evaluation.

Morphometrical investigation of medulloblastoma nuclei by S.A.M. (Shape Analytical Morphometry) software system.

In order to characterize medulloblastomas and to get over the difficulties sometimes encountered in differential diagnosis, a double morphometric procedure has been applied to its nuclei. The first consisted of size measurements (maximum diameter, area and perimeter), the latter is represented by S.A.M. (Shape Analytical Morphometry) software-system specifically implemented to describe shape of biological structure by analytical parameters. Analytical and dimensional parameters submitted to Hotelling's multivariate discriminant analysis gave the best results when used together in convenient discriminant subsets, thereby allowing a good distinction between medulloblastoma in comparison with neuroblastoma, Ewing's tumor, lymphoblastic and lymphocytic lymphoma. These results underline the usefulness of morphometric characterization also for practical diagnostic purposes.

Biological image analysis for microscopical diagnosis: the work-station S.A.M. (Shape Analytical Morphometry).

The S.A.M. (Shape Analytical Morphometry) software system and its related work-station give a generalized and easy-to-handle tool to face a classic and intriguing problem in biomedical morphological diagnosis. What is the shape of an object in a microscopic image? How can we understand the relationship between size and shape? According to Holloway: "Measurements such as length, width, height, whether in chords or arcs only describe space, ... and further run into the abyss of allometric correction ... if additional information (shape?) to size is expected, some method of allometric correction must be used." The S.A.M. software system assumes a logic architecture able to separate and to parametrize independently shape characteristics in terms of allometry and local pertubation by analytical procedures (polynomials, parabolic fitting, Fourier analysis) in addition to the classic evaluations of size and density carried out by image analyzers for microscopical diagnosis.

[Quantitative evaluation of the allometric transformation of of biological structures]. / Valutazione quantitativa delle transformazioni allometriche delle strutture biologiche.

Detection and quantification of allometry is a crucial problem in understanding morphological changes, both for systematic and morphogenetic purposes. A section of S.A.M. (Shape Analytical Morphometry) software system was used for this attempt. It consists of the following steps: a) boundary detection; b) starting point detection; c) size normalization; d) extraction of the fundamental shape by Kth order polynomials; e) finding of symmetry evaluator (S.A.E.) by means of a second degree equation. This last procedure gives an arc-chord complex that expresses a vector for allometry where intercept value was for application point, first degree coefficient was for direction and second degree coefficient was for modulus and versus. The main parameters, isometry fraction and allometry fraction may be understood referring them to morphogenetic models.

Non-Hodgkin's lymphoma diagnosis aided by the S.A.M. system.

In the attempt to discriminate between centroblasts and immunoblasts, an analytical morphometrical procedure was adopted, considering the nuclei of a randomly selected centroblastic and immunoblastic population from non-Hodgkin's lymphomas and the nucleoli of a more restricted number of the same neoplastic population. All the size-independent shape-descriptor parameters extracted for each step of the mathematical analyses used were submitted to Hotelling's multivariate discriminant analysis that enabled us to achieve a good distinction (3% error) between centroblasts and immunoblasts when nuclear and nucleolar parameters were used together.

Hand-mirror (HM) cells in acute lymphoblastic leukaemia (A.L.L.). An attempt at cellular classification by morphometric analytical parameters using the S.A.M. (Shape Analytical Morphometry) system.

A morphometric study of the nuclear and cytoplasmic shape of a blastic population in a case of HM-A.L.L. was performed by comparing the two differently shaped populations with and without HM configuration. The results obtained using analytical size-independent parameters created by the S.A.M. work-station enable us to characterize the shape of both blastic cell populations quantitatively, and strongly suggest the existence of shape modulation from one cellular type toward the other. Thus a possible sequence from blastic cells (having regular, rather round nuclei) to HM cells (characterized by high distortion of both nucleus and cytoplasm) was proposed.