Dibyajyoti Boruah

Morphometric study of microvessels in primary CNS tumors and its correlation with tumor types and grade

INTRODUCTION Alterations of microvasculature are integral to CNS neoplasia, and a diagnostic feature of high-grade gliomas. The objectives of this study were two fold: First, to correlate morphometrically measured microvessel density (MVD), microvessel caliber (VC), and percentage of total microvessel area (%TVA) with WHO histologic grade in various types of primary CNS tumors. Second, to evaluate if such a correlation could be further refined by using mathematical derivatives of measured parameters namely coefficient of variation of VC (COofVC), microvessel cross-sectional area (VCSA), and percentage of total VCSA (%TVCSA). MATERIALS AND METHODS Various microvessel parameters were assessed in a variety of 30 primary CNS tumors as consecutively encountered in routine surgical pathology practice including gliomas, meningiomas and others by image morphometry using CD34-immunostained sections. We introduced a novel method of effectively determining VC. Results were correlated with tumor type and grade. Appropriate statistical analysis was performed. RESULTS Microvessel characteristics, especially VC (p<0.0022), VCSA (p<0.0164), CVofVC (p<0.0001), %TVCSA (p<0.0002) and %TVA (p<0.0003) of tumors were significantly greater than normal tissue. MVD increased in all tumors, excepting meningiomas, and was significantly higher in gliomas (p<0.0062). MVD showed negative correlation with VC (r=-0.808) and VCSA (r=-0.848) in the normal brain but was less significant in tumors. Unlike tumors, caliber distribution of microvessels in normal brain was noted to follow a Gaussian pattern. Histological grades of tumors showed positive correlation with MVD (r=0.547), VC (r=0.606), CVofVC (r=0.623), VCSA (r=0.485), %TVCSA (r=0.783) and %TVA (r=0.603). Calculated scores, estimated from multiple regressions of vessel parameters, correlated well with histological grade, with S2 (calculated using all measured as well as mathematically derived microvessel parameters) being better than S1 (calculated using measured parameters: MVD and VC). CONCLUSION Tumor grades positively correlated with all microvessel parameters, with %TVCSA displaying the best. The correlation of %TVA with tumor grade was weaker than %TVCSA mainly due to the impact of MVD. These findings emphasize the value of VC as effectively measured using our novel method and best illustrated by its derivative %TVCSA (an indicator of blood flow), in addition to the well-recognized value of MVD in tumor prognostication. Multiple regressions of microvessel parameters provided the best correlation with grade. Morphometric analysis of microvessels in CNS tumor facilitates a better understanding of the tumor grade, tumor progression and overall prognosis.

Comparative evaluation of the marginal gingival epithelium in smokers and nonsmokers: a histomorphometric and immunohistochemical study.

Smoking is one of the strongest predictors of attachment and bone loss. Smokers demonstrate reduced inflammatory clinical signs, which could be due to local vasoconstriction and increased gingival epithelial thickness. The byproducts originating from tobacco oxidation modify the clinical characteristics and progression of periodontal disease. The aim of this study was to investigate the relationship between the thickness of marginal gingival oral epithelium, sulcular bleeding, and vascular caliber and density of the microvessels in smokers and nonsmokers with and without periodontitis and to better understand the role of smoking in relation to periodontal disease. One hundred twenty individuals were enrolled in this study and divided into four groups comprising 30 participants each. The clinical measurements carried out included probing depth, clinical attachment loss, and bleeding index, along with gingival biopsy specimens, which were subjected to immunohistochemical and histomorphometric analysis. Correlation of the clinical and histologic features revealed that smokers presented with fewer inflammatory signs, had fewer vascular elements in the subepithelial connective tissue layer, and showed a resultant increase in epithelial thickness irrespective of the presence of periodontitis. There was a mean increase of epithelial thickness of 181.3 μm (suprapapillary epithelial thickness [SET]) to 380.2 μm (maximal epithelial thickness [MET]) in smokers with periodontitis as compared to 157.4 μm (SET) to 325.3 μm (MET) in nonsmokers with periodontitis. The mean microvascular density in smokers with periodontitis was 325.4 per mm, which was found to be statistically significantly less than that of nonsmokers with periodontitis, who had a mean value of 412.13 per mm. The vessel caliber also was reduced in smokers, with a mean value ranging from 4.7 to 6.1 μm compared with a mean of 6.2 to 9.2 μm in nonsmokers, irrespective of the presence of periodontitis. Statistically significant differences were found in vascular density and thickness of gingival epithelium between smokers and nonsmokers with and without periodontitis. These differences may impact the progression of periodontal disease.

Correlation of microvessel parameters in invasive ductal carcinoma of the breast and fibroadenomas: a morphometric study

Modifications of microvascular configuration are essential features encountered during the progression of breast tumors. Our objectives were to correlate morphometrically evaluated microvessel parameters (microvessel density [MVD], microvessel caliber [VC], microvessel cross-sectional area [VCSA], percentage of total VCSA [%TVCSA], and total microvessel boundary density [TVBD]) with histologic grades of invasive ductal carcinoma (IDC) of the breast and benign breast lesions. Sixty cases of IDC presented with modified radical mastectomy, and 20 benign breast fibroadenomas were evaluated for various microvessel parameters, using CD34-immunostained histologic sections by computerized image morphometry. Samples were divided into 4 histologic groups: benign, grade 1, grade 2, and grade 3; mean with SD and range was evaluated for each group. Histologic grades showed a strong positive correlation with %TVCSA (ρ=0.773) and TVBD (ρ=0.811) and a moderate positive correlation with MVD (ρ=0.607), VC (ρ=0.609), and VCSA (ρ=0.616) when analyzed for all samples of the 4 groups. Except MVD, all parameters including age was the lowest (P<.001) for the benign group. Among the IDCs, differences of mean VC and VCSA were not significant; MVD, %TVCSA, and TVBD were the lowest in grade 1 and the highest in grade 3. Upper cutoff value of benign lesions for MVD was 155mm-2; VC, 9.94μm; VCSA, 94.42 μm2; %TVCSA, 1.33; and TVBD, 4.37mm-1. Total microvessel boundary density included the information of microvessel concentration and size showed the best correlation with grades. Microvessel density showed a positive correlation with grades in the IDCs, but for the differentiation of benign from malignant, VC, VCSA, %TVCSA, and TVBD showed excellent area under the receiver operating characteristic curve (area under the curve > 0.990), unlike MVD (area under the curve = 0.797).

Morphometric study of nuclei and microvessels in gliomas and its correlation with grades

INTRODUCTION Modifications of nuclear morphology in conjunction with alteration in microvascular configuration are essential features encountered during the progression of glial tumors. In order to gain more insight into tumor biology of gliomas, objectives of the study were selected (a) to correlate morphometrically evaluated nuclear parameters [nuclear area (NA), nuclear perimeter (NP), nuclear density (ND), percentage of total nuclear area (%TNA)] and microvessel parameters [microvessel density (MVD), microvessel caliber (VC), microvessel cross sectional area (VCSA), total microvessel boundary density (TVBD), percentage of total VCSA (%TVCSA)] with WHO grading; (b) extend such correlations to the ratio parameters: ratio of MVD to ND (MDV/ND), ratio of TVBD to %TNA (TVBD/%TNA) and ratio of %TVCSA to %TNA (%TVCSA/%TNA); and (c) to correlate microvessel and ratio parameters with NP and ND. MATERIALS AND METHODS A total of thirty gliomas managed at this institute during 2009-2012 were evaluated for various nuclear and microvessel parameters by image morphometry using a computerized digital photomicrograph system. For assessment of microvessel parameters CD34-immunostained sections were used while nuclear morphometry was performed on routine hematoxylin-eosin-stained sections. Appropriate statistical analysis was performed in correlation studies. RESULTS All nuclear morphometric parameters showed strong positive correlation with tumor grades (r>0.7). In contrast, though all microvessel parameters exhibited positive correlation with grades, the parameters TVBD and %TVCSA showed strong positive correlation. The ratio parameters (MVD/ND) and (TVBD/%TNA) showed negative correlation with grades, whereas (%TVCSA/%TNA) did not exhibit meaningful correlation with grades. Further, while all microvessel parameters showed positive correlation with NP and ND; ratio parameters showed negative correlation with them. CONCLUSION This study indicates that the parameters related to tumor growth (NA, NP, ND, %TNA), and angiogenesis showed increasing trend with tumor grades simultaneously; whereas the parameters related to supply of nutrients per nucleus showed decreasing trends with tumor grades, nuclear size and nuclear density. Thus, the former accounts for increased cellularity, mitosis, and vascular proliferation, while the latter culminates in tumor necrosis, all of which are essential components for grading of gliomas. The present study will therefore have a vital role as surrogate markers of grading of tumor.

Utility of Nuclear Morphometry in Predicting Grades of Diffusely Infiltrating Gliomas

Introduction. The ability to reliably differentiate neoplastic from nonneoplastic specimen and ascertain the tumour grade of diffusely infiltrating gliomas (DIGs) is often challenging. Aims and Objective. To evaluate utility of image morphometry in identifying DIG areas and to predict tumour grade. Materials and Methods. Image morphometry was used to analyze the following nuclear features of 30 DIGs and 10 controls (CG): major axis of nucleus (MAJX), minor axis of nucleus (MINX), nuclear area (NA), nuclear perimeter (NP), nuclear roundness (NR), nuclear density (ND), and percentage of total nuclear area (%TNA). Results. Statistically significant differences in all parameters, except NR, were observed between all groups, with strong positive correlation with tumour grade (r > 0.7). The mean values were maximum for HGG and minimum for CG. For NR, the difference between CG/HGG was statistically significant, unlike CG/LGG and LGG/HGG. It was observed that NA distributions for CG were nearly Gaussian type with smaller range, while gliomas displayed erratic pattern with larger range. NA and NP exhibited strong positive correlation with ND. Conclusion. Image morphometry has immense potential in being a powerful tool to distinguish normal from neoplastic tissue and also to differentiate LGG from HGG cases, especially in tiny stereotactic biopsies.

A morphometric and immunohistochemical study of melanocytes in periorbital hyperpigmentation

BACKGROUND An increase in number of melanocytes in the basal cell layer of the epidermis is an important feature in many disorders of hyperpigmentation. In this study, we attempted an objective evaluation of the linear density of melanocytes and keratinocytes, along with other epidermal characteristics, in periorbital hyperpigmentation using immunohistochemistry and morphometric techniques. METHODS Melanocytes and epidermal parameters were assessed by digital morphometry in 30 newly diagnosed cases of periorbital hyperpigmentation and 14 controls from the post-auricular region. Melanocytes were labelled with the immunohistochemical stains, Melan-A and tyrosinase. We studied the linear keratinocyte density, mean linear melanocyte density, ratio of melanocytes to keratinocytes, the ratio between inner and outer epidermal length, maximum epidermal thickness and minimum epidermal thickness. RESULTS Melan-A expression of melanocytes showed strong positive correlation (r=0.883) with the tyrosinase expression. Mean linear melanocyte density was 24/mm (range: 13-30/mm) in cases and 17/mm (13-21/mm) in controls and this difference was statistically significant (P<0.001). The mean ratio of melanocyte to keratinocyte was 0.22 (0.12-0.29) in cases and 0.16 (0.12-0.21) in controls; again, this difference was statistically significant (P<0.001). There was a mild negative correlation with linear keratinocyte density (r=-0.302) and the ratio between inner and outer epidermal length (r=-0.456). However, there were no differences in epidermal thicknesses. LIMITATIONS There were fewer control biopsies than optimal, and they were not taken from the uninvolved periorbital region. CONCLUSION Mean linear melanocyte density and the ratio of melanocytes to keratinocytes is increased in cases with periorbital hyperpigmentation. It is, therefore, likely that increased melanocyte density may be the key factor in the pathogenesis of periorbital hyperpigmentation.