Nuclear fractal dimension as a prognostic factor in oral squamous cell carcinoma.

Strong theoretical reasons exist for using fractal geometry in measurements of natural objects, including most objects studied in pathology. Indeed, fractal dimension provides a more precise and theoretically more appropriate approximation of their structure properties and especially their shape complexity. The aim of our study was to evaluate the nuclear fractal dimension (FD) in tissue specimens from patients with oral cavity carcinomas in order to assess its potential value as prognostic factor. Relationships between FD and other factors including clinicopathologic characteristics were also investigated. Histological sections from 48 oral squamous cell carcinomas as well as from 17 non-malignant mucosa specimens were stained with Hematoxylin-Eosin for pathological examination and with Feulgen for nuclear complexity evaluation. The sections were evaluated by image analysis using fractal analysis software to quantify nuclear FD by the box-counting method. Carcinomas presented higher mean values of FD compared to normal mucosa. Well differentiated neoplasms had lower FD values than poorly differentiated ones. FD was significantly correlated with the nuclear size. Patients with FD lower than the median value of the sample had statistically significant higher survival rates. Within the sample of patients studied, FD was proved to be an independent prognostic factor of survival in oral cancer patients. In addition this study provides evidence that there are several statistically significant correlations between FD and other morphometric characteristics or clinicopathologic factors in oral squamous cell carcinomas.

Hypoxia-inducible factor 1alpha/vascular endothelial growth factor axis in astrocytomas. Associations with microvessel morphometry, proliferation and prognosis.

Hypoxia-inducible factor (HIF)-1alpha is a transcription factor that promotes ischaemia-driven angiogenesis. The aim of this study was to determine the relation of HIF-1alpha to vascular endothelial growth factor (VEGF; an important angiogenic molecule in brain tumours), p53 expression, angiogenesis, proliferative potential and clinical outcome in a large series of diffuse astrocytomas. Expression of HIF-1alpha, VEGF, Ki-67 (a proliferation-associated marker) and p53 was determined immunohistochemically in 83 adult patients with supratentorial diffuse astrocytomas. Microvessels, highlighted by means of anti-CD34 immunohistochemistry, were enumerated with computer-assisted image analysis. Although HIF-1alpha and VEGF were expressed in the majority of cases, their levels increased significantly with increasing grade and proliferative potential. HIF-1alpha positively correlated with microvessel counts and VEGF with total vascular area and the presence of rounder vessel sections. There was a positive correlation of VEGF with p53 expression in astrocytomas and anaplastic astrocytomas. In univariate analysis, both VEGF and HIF-1alpha were associated with shortened survival in the entire cohort, but lost significance when grades II/III and grade IV were analysed separately. Multivariate analysis revealed that the combination of HIF-1alpha with grade was a significant prognostic indicator. HIF-1alpha expression may be used to refine the prognostic information provided by grade in patients with diffuse astrocytomas. Its adverse prognostic effect is most likely mediated by hypoxia, the driving force for HIF-1alpha accumulation.

The frequency and distribution of mast cells in pleomorphic adenomas of salivary glands.

AIM: To investigate whether the frequency and distribution of mast cells (MCs) in pleomorphic adenomas (PAs) of major and minor salivary glands justifies the suggestion that there exists an association between MCs and mucoid stromal changes in PAs. METHODS: The material consisted of 22 cases of pleomorphic adenoma (eight arising in major and 14 in minor salivary glands) and a control group represented by five cases of monomorphic adenoma (MA). Representative 3-microm thick, paraffin-embedded sections were stained with H&E and Azur A. Computer-aided image analysis was performed in order to evaluate the relative surface area occupied by epithelial and connective tissue components, as well as the absolute number of MCs. RESULTS: According to our findings, PAs from minor salivary glands contain significantly greater numbers of mast cells compared with tumours from major glands. Additionally, the distribution of MCs within the stromal connective tissue appeared not to be random. CONCLUSION: It is possible that differences in the pattern of connective tissue might influence the actual concentration of MCs and that these differences are responsible for the observed variations between major and minor gland PAs.

Clinicopathologic correlations of bone marrow angiogenesis in chronic myeloid leukemia: a morphometric study.

Various morphometric characteristics of microvessels, highlighted by means of anti-CD34 immunohistochemical staining, were evaluated in the bone marrow of 52 patients with chronic myeloid leukemia (CML) in chronic phase, in relation to several clinicopathologic parameters. Twenty control bone marrows and 15 cases of CML in blastic phase were also studied. Microvessel density (MVD), total vascular area (TVA) and several size- and shape-related parameters were quantitated in the region of most intense vascularization using image analysis. Overall, the group of chronic phase CML had higher MVD and size-related parameters and more branching microvessels than controls. Blastic phase was characterized by increased numbers of microvessels with a rounder shape and smaller caliber than chronic phase. A positive correlation emerged between marrow fibrosis and MVD as well as between white blood cell counts and rounder vessel sections. No relationship existed between microvascular parameters and Hasford or Sokal prognostic scores. In univariate analysis, overall and progression-free survival were adversely affected by MVD, size-related parameters, increased platelet count, age and spleen size. Multivariate analysis indicated that microvessel area was related to progression-free survival, whereas both MVD and area were significant prognosticators of overall survival, even when Hasford or Sokal scores are introduced into the model. Our data suggest that changes in angiogenic parameters may participate in the conversion of normal marrow to CML and ultimately to blastic transformation. More importantly, MVD and microvessel caliber are significant predictors of patient survival and progression.

Prognostic implications of microvessel morphometry in diffuse astrocytic neoplasms.

Astrocytic brain tumours, particularly malignant astrocytomas, are recognized to be highly vascular neoplasms with potent angiogenic activity. Recent research has shown that quantification of microvessel density (MVD), as a measure of the degree of angiogenesis, constitutes a strong prognostic indicator in patients with astrocytomas. However, the significance of other morphometric aspects of microvessel network has not been tested so far. In this report, histological sections from 70 astrocytomas (grades II to IV), immunostained for CD34, were evaluated by image analysis for the quantification of MVD, total vascular area (TVA), and microvascular branching, as well as several morphometric parameters related to vessel size or shape. Minor axis length increased with grade (P = 0.045) but MVD and TVA presented a peak in grade III (P = 0.033 and P < 0.001, respectively). Size and shape related parameters affected survival in univariate analysis of grade IV and grades II/III, respectively. In multivariate analysis, only branching counts, along with age and grade, were the independent predictors of survival. Although MVD, TVA and branching counts were adversely related to disease-free survival in grades II and III (univariate analysis), only TVA remained statistically significant in multivariate analysis. It is concluded that TVA and branching counts are prognostically more informative than MVD for patients with diffuse astrocytic tumours.

Prognostic evaluation of the microvascular network in myelodysplastic syndromes.

Considering the recently stated suggestion of neovascularization being implicated in myelodysplastic syndromes (MDS) pathogenesis, we evaluated multiple morphometric microvascular characteristics in MDS, in relation to clinicopathologic factors and prognosis. Trephines from 50 newly diagnosed MDS patients were immunostained for factor VIII and compared to those from 20 controls, 10 chronic myelomonocytic leukemia (CMML) and 12 acute myeloid leukemia (AML) patients. Quantitation of microvessel density (MVD), area, total vascular area (TVA), major and minor axis length, perimeter, compactness, shape factor, Feret diameter, and the number of branching vessels was performed by image analysis. Overall, the MDS group had significantly higher MVD, TVA, minor axis and shape factor values and significantly lower compactness than the control group. AML was characterized by increased vascularity compared to MDS and CMML, as well as by the presence of flattened microvessels (lower values of shape factor). Hypercellular MDS showed higher MVD. RA/RARS displayed larger caliber vessels than RAEB, which explains the favorable prognostic effect of increased size-related parameters on progression and/or survival. Moreover, decreased compactness and MVD were independent predictors of longer progression-free survival. It is concluded that angiogenesis is involved in the conversion of normal marrow to MDS and ultimately to AML and that disease progression within MDS is accompanied by qualitative alterations of the microvascular network. Furthermore, size-related parameters affect survival, while shape-related parameters and MVD are more influential with regard to progression-free survival.

Morphometric microvascular characteristics predict prognosis in superficial and invasive bladder cancer.

Recent research has shown that neovascularization, quantitated by microvessel density (MVD), constitutes a strong prognostic indicator in patients with invasive urothelial carcinomas. These studies, however, have focused only on MVD as the only factor reflecting angiogenesis in transitional-cell carcinomas (TCCs). The objective of this report was to evaluate multiple morphometric microvascular characteristics besides MVD in superficial and muscle-invasive TCCs separately, to provide a better approach to the relationship between angiogenesis, clinicopathological parameters, and prognosis. Histologic sections from 115 TCCs [35 superficial (T1) and 80 muscle-invasive] were immunostained for CD31 and evaluated using image analysis for the quantitation of MVD, area, total vascular area, major axis length, minor axis length, perimeter, compactness, shape factor, and Feret diameter. Patients were followed-up until death (n=31) or for an average of 42.2 months (median 38.5 months). MVD increased with progressing T category (P=0.049) but area (P=0.033), major axis length (P=0.022), perimeter (P=0.043), and Feret diameter (P=0.042) were highest in T2 tumors. Area was the single independent predictor of adverse significance in T1 TCCs, whereas for muscle-invasive tumors, survival was independently predicted by MVD. Regarding disease-free survival in superficial tumors, the single significant independent parameter was compactness, whereas area was an independent favorable indicator of disease-free survival for patients with invasive TCCs. It is concluded that the prognostic significance of neovascularization is better assessed by area and shape-related morphometric characteristics, whereas MVD becomes influential only with regard to overall survival of patients with invasive tumors.

Nuclear/Nucleolar morphometry and DNA image cytometry as a combined diagnostic tool in pathology of prostatic carcinoma.

Paraffin tissue sections from 50 patients with prostate adenocarcinoma were used to study nuclear and nucleolar morphometric features by image analysis. The results were compared to DNA ploidy and Gleason grade. In the examined histological samples nuclear and nucleolar areas were positively interrelated. It was also noticed that the higher the percentage of nucleolated nuclei, the bigger the nuclear and nucleolar areas. The morphometric characteristics did not differ significantly among the four grades of the examined specimens. In well-differentiated carcinomas the DNA index was lower than in the rest at a statistically significant level. Hypodiploid carcinomas were found to possess significantly bigger nuclear areas than any other DNA index group. Morphonuclear evidence of anaplasia and DNA aneuploidy may be used as diagnostic tools in prostate cancer in addition to Gleason grade.

Membranous glomerulonephritis: a morphometric study.

Archival material from 45 renal biopsies with a diagnosis of idiopathic membranous glomerulonephritis (MGN) were studied by computer-aided image analysis in order to evaluate the prognostic significance of glomerular and interstitial morphometry in MGN. The control group consisted of thirty seven normal renal biopsy specimens. The surface area, the perimeter, the major axis length and the shape factor of renal glomeruli as well as the percentage of the interstitial fibrosis were measured. All the morphometric parameters related to the size of glomeruli had significantly higher values in the patient group (p = 0.000 for all the parameters). However, no significant difference of the glomerular size between different stages of MGN was observed. In contrast, the percentage of interstitial fibrosis increased as the MGN stage rose (median values: 10.3% in stage 1, 14.2% in stage II, 26.9% in stage III, 28.9% in stage IV and 34.2% in stage V, Kruskal-Wallis ANOVA H = 37.645, p = 0.000). In the multivariate analysis the percentage of interstitial fibrosis was the only independent prognostic factor (p = 0.013). Our findings suggest that, in membraneous glomerulonephritis, the interstitial fibrosis increases as the MGN stage progresses, while the size of renal glomeruli has increased at a very early stage of the disease. This fact may indicate that interstitial fibrosis, not glomerular lesions, is mainly responsible for the reduction of renal function.

Renal leiomyosarcoma: report of three cases and review of the literature.

In the present study three cases of renal leiomyosarcoma are presented. All the patients were complaint about pain at right or left lumbar area and microscopic or gross haematuria. The clinical examination did not confirm any pathologic finding. The ultrasonographic and CT scan investigation of the patients revealed a renal mass. Under the frozen section diagnosis of malignant tumor in two cases and the cytological diagnosis of renal cell carcinoma in the third case, a radical nephrectomy was performed. In all the cases the pathological diagnosis was renal leiomyosarcoma. Because of the rarity of this neoplasm, the literature is reviewed, presenting the symptoms, the radiological findings, the diagnostic criteria and the differential diagnosis of the tumor. Radical nephrectomy remains the treatment of choice for this tumor, which exhibits an aggressive biological behavior and an unfavorable prognosis.

Association of single umbilical artery with congenital malformations of vascular etiology.

The possible association of a single umbilical artery (SUA) with malformations of vascular etiology is investigated in this study. Four hundred twelve fetal and embryonic autopsies showing one or more congenital malformations, collected over 7 years, were reviewed. Microscopic confirmation of a SUA was evident in 20 cases (4.85%). The two subgroups with 2 (nA = 20) or 3 umbilical vessels (nB = 392) were compared with each other, in relation to the frequency of malformations per organ system. In the group of fetuses with congenital malformations, no association was observed between SUA and the incidence of CNS, cardiac, pulmonary, or genital malformations. However, there was a significantly higher incidence of atresia of hollow organs (P = 0.003), renal aplasia (P = 0.034), and limb reduction defects (LRD) (P = 0.0383) when only a single umbilical artery was present. This suggests a possible etiopathogenetic association of SUA with congenital malformations of vascular etiology. Furthermore, the findings of our study suggest that prenatal identification of a SUA warrants a thorough search for atresias, renal aplasia, and LRD-type malformations.

A morphometric study of neovascularization in colorectal carcinoma.

BACKGROUND: Neovascularization reportedly is correlated with metastasis, recurrence, and prognosis in some types of tumors. Microvessel quantification in so-called "hot spots" has been studied extensively as the only factor reflecting angiogenesis in various malignant tumors. The objective of this report was to evaluate multiple morphometric microvascular characteristics in addition to microvessel density (MVD) in colorectal carcinomas to provide a better approach to examining the relation between angiogenesis and clinicopathologic factors and prognosis. METHODS: Histologic sections from 106 colorectal adenocarcinomas and 17 adenomas, immunostained for factor VIII, were evaluated by image analysis for the quantification of MVD, total vascular area (TVA), and microvascular branching, as well as several morphometric parameters related to the vessel size or shape. RESULTS: MVD gradually decreased with progressing Dukes stage. The vascular branching counts were significantly higher in carcinomas than in adenomas, and remained unaffected through progressing Dukes stages. Shape-related parameters showed significant differences between carcinomas and adenomas and between different grades of differentiation. Branching counts and TVA were the only factors found to be of prognostic significance. CONCLUSIONS: This study provides evidence that neovascularization is an early critical event in colorectal tumorigenesis, reaching a maximum level early in the malignant process. Its prognostic significance is better assessed by quantification of TVA and the branching pattern of microvessels, whereas MVD does not provide significant prognostic information for colorectal carcinoma patients.

Cell proliferation rate and nuclear morphometry in Roberts syndrome.

Roberts syndrome (RS) is a rare autosomal recessive disorder characterized primarily by symmetric reduction anomalies of all limbs, growth retardation and craniofacial abnormalities. Most RS patients are reported to present a typical abnormality of their constitutive heterochromatin, accompanied by abnormal cytological growth characteristics. We present an extremely severe case of an RS fetus, karyotypically documented, with a clinical presentation including growth deficiency, tetraphocomelia, frontal meningocele, craniofacial abnormalities and penile enlargement with hypospadias. Nuclear morphometrical analysis in tissues of various organs revealed a reduced nuclear size in RS as compared to normal controls, and statistically significant differences in morphometric parameters related to the nuclear shape. Immunohistochemical study of the same organs showed a reduced expression of proliferating cell nuclear antigen in the presented case, thus indicating a decreased cell proliferation rate in RS. Our results reconfirm previously reported findings in cultured fibroblasts of RS cases, thereby reinforcing on a histologic level, the hypothesis that reduced cell proliferation may be involved in the growth retardation and the reduction abnormalities observed in RS.

Expression of apoptotic and proliferation markers in meningiomas.

Fifty-two intracranial, totally excised meningiomas were immunohistochemically analysed for the expression of bcl-2 and p53 proteins, in parallel with the assessment of the proliferating cell nuclear antigen labelling index (PCNA LI) and the mitotic index (MI). bcl-2 was expressed in 26.8 per cent and p53 in 32.6 per cent of the tumours, exhibiting a similar staining pattern, with low levels of immunoreactive cells. The bcl-2-positive/p53-negative subgroup showed a significant association with a benign histological pattern. Expression of bcl-2 appeared to have no influence on the rate of recurrence; p53 expression was the only factor with prognostic significance for recurrence (p = 0.10). There was no interaction between bcl-2 and p53 expression. The PCNA LI was correlated with the MI and the grade of malignancy, proving to be a useful proliferation marker and an additional indicator of the more anaplastic histological patterns in meningiomas. Proliferation indices appeared to have no correlation with the recurrence rate of totally resected tumours. Meningiomas expressing the bcl-2 protein presented a high proportion of proliferating cells in S phase. In contrast, all the tumours which recurred had a minimal S-fraction of proliferating nuclei. These findings may improve our understanding of the interaction between cell proliferation, expression of apoptotic markers, and recurrence in meningiomas.

Fetal akinesia deformation sequence in a highly developed acardius twin.

We report a case of a holoacardius twin with extremely advanced development of the head, face, upper and lower limbs in the absence of all thoracic and upper abdominal viscera and associated with intestinal and anal atresia. The malformed fetus also had craniofacial abnormalities, hydrops, cystic hygroma of the neck, arthrogryposis and pterygia. The monozygous co-twin was found to be normal. The association of acardia with the typical characteristics of the fetal akinesia deformation sequence has not been previously described in the literature.

Prenatal ultrasonographic detection of the tracheal atresia sequence.

We report a rare case of tracheal atresia, without fistula, in a 19 weeks' female fetus. The abnormality was detected by prenatal ultrasonography, which demonstrated a bilateral and uniform hyperechogenicity and enlargement of the lungs, associated with ascites and moderate oligohydramnios. We suggest that oligohydramnios may be helpful in distinguishing the upper respiratory tract obstruction from congenital cystic adenomatoid malformation of the lungs, type III. A morphometric study revealed a three-fold more dense pulmonary vasculature in comparison with two control cases of the same gestational age. The pathological finding of pulmonary and vascular hyperplasia as well as the pathophysiology of the syndrome are discussed.

Computerized determination of proliferating cell nuclear antigen expression in meningiomas. A comparison with non-automated method.

Proliferating cell nuclear antigen (PCNA) expression has been proven to be a significant marker of cell proliferation in meningiomas, which correlates with growth rate and, as shown by several authors, possibly provides prognostic information concerning biologic behavior. However, the current method for determining PCNA labeling index (LI) is tedious and time consuming like all the nonautomated methods for evaluating cell kinetics, presenting high interobserver and interlaboratory variability and low reproducibility. In the present study, we introduce a semi-automated computer-assisted image analysis method for determining PCNA LI in 38 meningiomas, in parallel with the current nonautomated method. Image analysis technique permits unbiased cell counting, standardizes the degree of staining intensity and provides instant results. By calculating coefficient of variability, the method proved to be highly reproducible. The correlation between the results provided by the nonautomated and the semiautomated image analysis method showed a high agreement between them, with a correlation coefficient, r, of 0.82. In conclusion, we consider that image analysis contributes to the accuracy, reproducibility, and practicality of PCNA LI determination so that along with other useful parameters this significant marker may serve to predict the clinical behavior in meningiomas.

Subacute necrotizing encephalomyelopathy (Leigh's disease): a clinicopathologic study of ten cases.

Archival material and clinical data of 10 autopsy cases of Leigh's disease (LS), aged from 44 days to 9 years at death, were reviewed. Development delay, irregular respiration, feeding difficulty, and abnormal eye signs were the most common symptoms. Seizures (five of ten cases) were also frequent. In most patients, the diagnosis of LS was established postmortemly by the presence of symmetrical spongiform lesions affecting several brain centers at autopsy. The histologic examination disclosed associated hypertrophic cardiomyopathy in six cases, while fatty infiltration of the hepatocytes was observed in four cases. Microvesicular degeneration of the renal tubular epithelial cells was also seen in four cases. Our observations suggest that liver and kidney involvement is a component of LS and that this rare entity has to be considered as a polysystematic disorder, able to affect other organs besides the nervous system and the heart, a fact which has not been emphasized enough in the existing literature.

Computerized nuclear morphometry of renal cell carcinomas.

The aim of this study was the evaluation of the usefulness of nuclear morphometry in the pathology of renal cell carcinoma by computer-aided image analysis and the statistical comparison of nuclear morphometric parameters with the histologic grade of the tumors. 44 cases of renal cell carcinoma, classified by two independent pathologists into 4 histologic grades (I-IV), were examined. The following 5 nuclear morphometric parameters were measured in a large number of randomly selected nuclei of each case: Major axis length, area, elongation, roundness and compactness. The statistical evaluation was performed using one-way analysis of variance between the four groups of the histologic grades (I-IV). Between them, there was a statistically very significant difference of the mean value of all the evaluated parameters. The values of the estimated parameters, with the exception of roundness and compactness, showed a strong tendency to increase in proportion to histologic grade. Our results suggest that image analysis is a reproducible and objective method for the grading of renal cell carcinoma, and it can be helpful in the unbiased evaluation of such tumors.