Computer-assisted Quantitative Image Analysis Research Articles

Histomorphological and Morphometric Evaluation of Microvessel Density in Nodal Non-Hodgkin Lymphoma Using CD34 and CD105.

Background Expression of angiogenic markers determined by microvessel density (MVD) could be used as a reliable predictor of prognosis and as a potential target for antiangiogenic therapy in different categories of non-Hodgkin lymphoma (NHL). Aims The aim of this study was to evaluate MVD using immunohistochemical methods and computer-assisted quantitative image analysis in nodal NHL patients and compare CD34 and CD105 expression in lymph nodes of NHL patients. Materials and Methods The present study was conducted on 60 lymph node biopsies received in the Department of Pathology at our tertiary care center for histopathological examination. Representative paraffin-embedded tissue sections were stained with hematoxylin and eosin along with immunohistochemical stains for CD34 and CD105. MVDs were analyzed at 400× using automated image analyzer by two investigators independently. Statistical Analysis Data were calculated, tabulated, and statistically analyzed using SPSS (Statistical Package for Social Studies) statistical program version 18. The values entered were mean of morphometric parameters. In all tests, p -values below 0.05 were regarded as significant. Results MVD was determined by CD34 and CD105 antibody highly correlated with different categories of NHL. Higher MVD was observed in cases of aggressive NHL as compared with indolent NHL and the difference was statistically significantly. MVD using CD105 was correlated more strongly as compared to CD34 with different categories of NHL. Conclusion The present study concluded that NHL exhibits potent angiogenic activity that increased significantly with increasing aggressiveness. The study also demonstrated that CD105 is more specific than CD34 as a marker of neoangiogenesis in NHL.

Pathobiology of the 129:Stat1\u2212/\u2212 mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype

BackgroundStat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1−/− host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors.MethodsMice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1−/−-derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells.ResultsTumorigenesis in 129:Stat1−/− originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1−/− tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor.Conclusions129:Stat1−/− is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Significance of vascular endothelial growth factor and CD31 and morphometric analysis of microvessel density by CD31 receptor expression as an adjuvant tool in diagnosis of psoriatic lesions of skin.

Pathogenesis of psoriasis is a debated issue. Several mechanisms have been proposed to identify the etiology and pathogenesis so that specific treatments can be given to patients with psoriasis. (1) To compare pattern and distribution of vascular endothelial growth factor (VEGF) and CD31 in patients with psoriasis and other psoriasiform lesions of skin. (2) To study the correlation between VEGF and CD31 expression, clinical severity, and histopathology of psoriasiform lesions of skin. (3) Evaluation of microvessel density (MVD) by using computer-assisted quantitative image analysis in psoriatic skin lesions. This study was conducted on eighty cases, out of which forty were diagnosed cases of psoriasis and forty cases of clinically suspected psoriasiform lesions, submitted in the Department of Pathology, Pt. B.D. Sharma, University of Health Sciences, Rohtak, for histopathological examination. Histopathological sections were stained by routine hematoxylin and eosin staining, and these biopsies were further subjected to immunohistochemical staining with VEGF and CD31 as per standard technique. Assessment of various histopathological features revealed strong correlation between epidermal hyperplasia, suprapapillary thinning, and elongation of rete ridges. Suprabasilar keratinocytes in psoriatic lesions stained intensely for VEGF. The difference for number of microvessels and MVD in psoriasis and psoriasiform lesions was statistically significant. Correlation between intensity of VEGF staining by suprabasilar keratinocytes and MVD was found to be highly significant in psoriatic lesions. The present study concluded that psoriatic lesions exhibit potent angiogenic activity. Early lesions show increased MVD along with other histomorphological parameters such as hypogranulosis, parakeratosis and Munro's microabscesses. Overexpression of VEGF by suprabasilar keratinocytes correlated with increased MVD in papillary dermis.

Inhibition of corneal neovascularization after alkali burn: comparison of different doses of bevacizumab in monotherapy or associated with dexamethasone

To compare the effects of different doses of bevacizumab with both saline and dexamethasone on inflammatory angiogenesis in the rat cornea induced by small chemical lesions. Corneal chemical cauterization was performed on 24 rats. Animals were divided randomly into six groups and received a daily subconjunctival injection for 7 days of: balanced salt solution 0.1 mL or dexamethasone phosphate 4 mg/day or bevacizumab 2.5 mg/day, 3.75 mg/day, 5.0 mg/day or bevacizumab 5.0 mg/day + dexamethasone phosphate 4 mg/day. Clinical examination under slitlamp was performed daily for 7 days to evaluate corneal opacity and vessel size evolution. Computer assisted quantitative image analysis was used to measure the total corneal area covered by neovascularization. At final examination, the dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups showed a significant lowering in corneal opacity score as compared with control (P = 0.024, P = 0.006 and P = 0.013, respectively). Also, a significant reduction on new vessels size score was observed. Surface of corneal neovascularization was significantly reduced in dexamethasone, bevacizumab 5.0 mg/day and dexamethasone + bevacizumab groups compared with control (P =0.045, P = 0.047 and P = 0.044, respectively). Our study demonstrates the ability of a 5.0 mg/day bevacizumab subconjunctival injection, in monotherapy or associated with dexamethasone, to cause a short-term involution of corneal neovascularization after corneal alkali burn. Combination of both of these treatments may have advantages to monotherapy approaches.

Increased angiogenesis is associated with poor prognosis of squamous cell carcinoma of the vulva

Increased angiogenesis is associated with an increased risk of malignant transformation in many organs. The occurrence, characteristics and significance of angiogenesis in vulvar squamous cell carcinoma are less well known. In this study, vessel number as well as vessel size, vessel shape and vessel antibody staining intensity were determined in 23,091 vessels in CD34-stained histological specimens of 42 patients by computer-assisted quantitative image analysis methods and findings were related to tumor morphology, clinical characteristics and patient survival. The 2- and 5-year survival rates were 81.1 and 64.1%, respectively. High disease stage and high histological grade indicated poor prognosis. In an age-adjusted multivariate analysis of stage, grade and extent of invasion, these variables compiled a statistically significant prognostic factor (p=0.001). Tumor histopathological type was a prognostic factor in our study and keratinising tumors did significantly better. Large tumor size, but not depth of invasion, and spray type growth pattern also indicated poor survival. In an age-adjusted multivariate analysis of stage, grade and extent of invasion, these variables compiled a statistically significant prognostic factor (p=0.001), but none of these variables alone proved to be an independent prognostic factor. High vessel number and increased vessel size were also significant indicators of poor survival, as were vessel characteristics: vessel number, vessel size, vessel shape and staining intensity together. Increased angiogenesis and altered vessel characteristics were prognostic factors in determining patient survival in this study. Analysis of angiogenesis provided new information on tumor behaviour and provided markers for survival analysis in neoplasms classified by tumor type and growth pattern.

Involvement of neurotrophin‐4/5 in regeneration of the periodontal Ruffini endings at the early stage

Little is known about the role of neurotrophin-4/5 (NT-4/5) in the regeneration of mechanoreceptors. Therefore, the present study examined the regeneration process of Ruffini endings in the periodontal ligament in nt-4/5-deficient and wildtype mice following transection of the inferior alveolar nerve by immunohistochemistry for protein gene product 9.5 (PGP 9.5), a general neuronal marker, and by computer-assisted quantitative image analysis. Furthermore, rescue experiments by a continuous administration of recombinant NT-4/5 were performed and analyzed quantitatively. At postoperative day 3 (PO 3d), almost all PGP 9.5-positive neural elements had disappeared; they began to appear in both types of animals at PO 7d. At PO 10d, almost all nerve fibers showed a beaded appearance, with fewer ramifications in both types of mice. Although the regeneration proceeded in the wildtype, a major population of the periodontal Ruffini endings continued to display smooth outlines at PO 28d in the nt-4/5 homozygous mice. The reduction ratio of neural density reached a maximum at PO 3d, decreased at PO 10d, and later showed a plateau. In a rescue experiment, an administration of NT-4/5 showed an acceleration of nerve regeneration in the homozygous mice. These findings indicate that the nt-4/5-depletion causes a delay in the regeneration of the periodontal Ruffini endings, but the delay is shortened by an exogenous administration of NT-4/5. Combined with our previous findings of bdnf-deficient mice (Harada et al. [2003] Arch Histol Cytol 66:183-194), these morphological and numerical data suggest that multiple neurotrophins such as NT-4/5 and brain-derived neurotrophic factor (BDNF) play roles in their regeneration in a stage-specific manner.

Altered extent, pattern and characteristics of microvascular density are indicators of neoplastic progression in the endometrium

The occurrence, structure and extent of microvascular density were examined in normal endometria, hyperplasia of different types and adenocarcinomas of different degrees of differentiation to determine their biologic and clinical significance in tumor development and progression. Computer-assisted quantitative image analysis was carried out on 12,500 vessels in regard to vessel number, vessel volume, size, shape and extent of vessel antibody staining, with sensitivity and reproducibility exceeding 99%. The results showed the extent, pattern and characteristics of microvascular density to be intimately associated with extent of tumor development and degree of differentiation of the tumor. Vessel number increased with superficial location in normal endometrium, with increased degree of hyperplasia and atypia and with increased degree of dedifferentiation of adenocarcinoma. Increased vessel shape alterations were characteristic of atypical complex hyperplasia when compared to other types of hyperplasia. Vessel number, size and shape were similar in proliferative endometrium and simplex type hyperplasia, and microvascular density in atypical complex hyperplasia was similar to that in well-differentiated adenocarcinoma. The results indicated that vessel shape alterations occur during progression of hyperplasia and vessel size increase occurs in complex-type hyperplasia and in moderately differentiated adenocarcinomas. We conclude that microvascular density is associated with endometrial location and with specific patterns of alteration in different stages of endometrial disease. The results suggest potential clinical applications of vessel analysis for determination of clinical behavior of endometrial preneoplastic and neoplastic alterations.

Histopathological parameters determining lesion colours in the naevus of Ota: a morphometric study using computer-assisted image analysis.

Naevus of Ota manifests various colours ranging from light brown to blue. Naevus colours have been claimed to reflect the depth of melanin pigments but the claim has rarely been substantiated by quantitative studies. We attempted both quantitative and qualitative analyses of the naevus of Ota to find out relations between histological patterns or parameters of melanin/melanocytes and lesion colours. Lesion colours were determined by one of the authors and were confirmed by a separate panel of dermatologists. Forty biopsy specimens of naevus of Ota were evaluated by both computer-assisted quantitative image analysis and a previously proposed conventional pattern analysis. The mean area fraction (AFmean) of melanin, the depth of the maximum area fraction of melanin (level of AFmax) and the depth of the deepest infiltrating melanocyte were significantly greater or deeper for bluish lesions than brownish lesions. Based on the qualitative pattern analysis we found that all the brownish lesions demonstrated superficial dermal melanin pigments, whereas bluish lesions tended to show more heterogeneous histological patterns. Eyelid lesions, all of which were bluish, revealed greater AF(mean) value than cheek lesions, presenting as either brownish or bluish colours. Quantitative analysis indicated that pigment density measures such as AFmean could be as important as the depth of melanocytes in the explanation of the lesion colours in naevus of Ota. However, qualitative pattern analysis failed to link specific patterns with lesion colours, especially in bluish lesions, probably due to the lack of consideration of the pigment density.

A decrease in size and number of basal forebrain cholinergic neurons is paralleled by diminished hippocampal cholinergic innervation in mice lacking leukocyte common antigen-related protein tyrosine phosphatase activity

The leukocyte common antigen-related (LAR) receptor, composed of an extracellular region with three immunoglobulin-like and eight fibronectin type III-like domains, and a cytoplasmic region containing two protein tyrosine phosphatase domains, is thought to play a role in axonal outgrowth and guidance during neural development. LAR mutant mice were generated completely lacking the two cytoplasmic protein tyrosine phosphatase domains, resulting in the loss of ability to bind intracellular associating proteins, but (may be) still containing the ability to perform extracellular functions. A reduction in size of basal forebrain cholinergic neurons and diminished hippocampal innervation reported for knockout mice that contain a leaky gene trap inserted into the 5′ part of the LAR gene [Yeo T. T. et al. (1997) J. Neurosci. Res. 47, 348–360] warranted a computer-assisted quantitative image analysis throughout the basal forebrain and hippocampus of our LAR mutant mice. The total number, longest diameter and cell body area were calculated for the choline acetyltransferase-positive neurons in the medial septum and vertical diagonal band, and optical density measurements were performed to determine the extent of acetyl cholinesterase-positive fibre innervation of the different layers in the dentate gyrus. In LAR mutant mice, the number of cholinergic cells was significantly reduced (∼25%) in the vertical diagonal band. Also, the cross-sectional area of the cholinergic neurons in the medial septum and vertical diagonal band was reduced (5%). These findings were paralleled by a diminished cholinergic innervation of the supragranular (18%) and molecular (4%) layers of the dentate gyrus. Thus, LAR protein tyrosine phosphatase activity appears crucial for size, number and target projection of basal forebrain cholinergic neurons, further strengthening a role for LAR in CNS development.

Endpoint markers for cancer chemoprevention trials derived from the lesion of precancer (intraepithelial neoplasia) measured by computer-assisted quantitative image analysis.

Endpoint markers for cancer chemoprevention clinical trials are described that are developed from the morphological properties of the precancerous lesion of intraepithelial neoplasia itself, as measured by computer-assisted quantitative image analysis. The markers include increased proliferative fraction (percentage MIB-1 positive nuclear area); nuclear DNA content (DNA ploidy), including DNA content exceeding fivefold the haploid amount (5C-exceeding rate); nuclear/nucleolar morphometry; and disorganization of nuclear chromatin pattern as characterized by Markovian parameters and other functions. A significant new advance in image analysis is the process of "tiling," in which hundreds of full monitor image fields of a given histological section at x40 magnification are reduced in size and fused seamlessly to produce a single image of the histological section at x1.25 magnification. The operator may review the low-power image and retrieve x40 magnification of any desired area by point/clicking with a mouse.

Regular albuterol or nedocromil sodium — effects on airway subepithelial tenascin in asthma

Both albuterol and nedocromil sodium have been recognized to possess certain anti-inflammatory properties. However, there are no data on the impact of these drugs on the pathophysiology of the bronchial extracellular matrix in asthma characterized by enhanced tenascin (Tn) expression, known to occur proportional to the severity of asthma. This paper reports data from a morphometric study on the effects of regular treatment with inhaled albuterol or nedocromil sodium on the extent of bronchial subepithelial deposition of Tn, collagen types III, IV, and VII and mucosal infiltration with macrophages. Thirty-two patients (14 women) with chronic asthma, aged 38·7 years (median) with a median forced expiratory volume in 1 sec (FEV 1) of 74·4% predicted, were selected to undergo fibre-optic bronchoscopy with bronchial biopsies before and after 12 weeks of treatment with either inhaled albuterol 0·2 mg or nedocromil sodium 4 mg four times daily according to a double-blind protocol. Cryostat sections of the biopsy specimens were studied by indirect immunostaining techniques using monoclonal antibodies and computer-assisted quantitative image analysis. Albuterol treatment significantly reduced the median thickness of subepithelial Tn expression from 9·7 to 6·3 μm ( P=0·023) and macrophage numbers in the epithelium ( P=0·034), lamina propria ( P=0·039) and entire mucosa ( P=0·033), whereas nedocromil sodium had no effect. Expression of the collagen types was not affected by either treatment. There was no identifiable statistical difference between the two treatments for any of the outcome variables measured. Nevertheless, the results demonstrate that even a short-acting β 2-agonist may exert anti-inflammatory potential sufficient to interfere with the basic mechanisms of asthma as shown by reduction of subepithelial Tn content and mucosal macrophage count.

Properties of intraepithelial neoplasia relevant to cancer chemoprevention and to the development of surrogate end points for clinical trials.

Cancer chemoprevention is defined as the prevention of cancer by the administration of diet supplements or drugs. A drug discovery effort should therefore focus on finding agents that will avert the process of intraepithelial neoplasia which precedes invasive cancer. Over 30 agents developed by the chemoprevention program at the National Cancer Institute are being tested against intraepithelial neoplasia of many organ sites in more than 80 clinical trials. Two basic mechanisms underlie the onset and development of intraepithelial neoplasia. First is the development of the two precursor lesions of chronic diffuse epithelial hyperplasia and genomic instability, the latter being produced by "mutator" mutations in genes responsible for genomic stability, by gene copy amplification or loss from DNA breakage-fusion-anaphase-bridge cycles, by unequal sister chromatid exchange, and by accumulation of double minutes. Second is the development of multicentric intraepithelial neoplastic lesions which independently progress through each of the following processes at a continuously accelerating rate: clonal evolution, hyperproliferation, production of genomic structural variants, and apoptosis. Recommended chemoprevention strategies based on these mechanisms are (i) the development of better technology for early diagnosis, (ii) the development of multiple agents that block intralesional proliferation at steps along the signal pathway of mitotic signal transduction and along the signal pathway of synthesis of daughter cell components, (iii) the development of nontoxic anti-inflammatory agents, anitoxidants, antimutagens, and proapoptotics, (iv) the avoidance of "clonal escape" through use of drug combinations, and (v) the use of computer-assisted quantitative image analysis to assay modulation of surrogate end points in chemoprevention clinical trials.

Properties of intraepithelial neoplasia relevant to the development of cancer chemopreventive agents

Cancer chemoprevention is concerned with the development of drugs or diet supplements that will avert the onset or stop the progression of the intraepithelial neoplasia which precedes invasive cancer. Two basic processes underlie the onset and development of intraepithelial neoplasia. First is genomic instability (often associated with chronic diffuse epithelial hyperplasia), which is the increased production of genomic structural variants due to unrepaired DNA breaks with secondary formation of abnormal structures, including "mutator" mutations in genes responsible for genomic stability, gene copy amplification or loss from DNA breakage-fusion-anaphase bridge cycles, unequal sister chromatid exchange, and accumulation of double minutes. Second is the development within an epithelium having genomic instability of multicentric neoplastic lesions that independently progress through each of the following processes at a continuously accelerating rate: clonal evolution, hyperproliferation, production of genomic structural variants, and apoptosis. Recommended chemoprevention strategies based on these mechanisms are (1) early diagnosis and treatment of genomic instability before the appearance of intraepithelial neoplasia, i.e., during the "predysplastic" or "premorphologic" phase, (2) development of multiple agents that block intralesional proliferation at steps along the "command" pathways of mitotic signal transduction and along the "execute" pathways of synthesis of daughter cell components, (3) development of nontoxic antiinflammatory agents, antioxidants, antimutagens, and proapoptotics, (4) avoidance of "clonal escape" through use of drug combinations, and (5) use of computer-assisted quantitative image analysis to assay modulation of surrogate endpoints in chemoprevention clinical trials.

Biomarkers of Premalignant Breast Disease and Their Use as Surrogate Endpoints in Clinical Trials of Chemopreventive Agents

: Surrogate endpoint biomarkers (SEBs) are tissue changes observed in premalignant disease that highly correlate with cancer risk. They are urgently needed as endpoints in clinical trials of chemopreventive agents for breast premalignant disease because they require less time, money and effort compared to the conventional endpoint of cancer incidence reduction. The program of the Chemoprevention Branch, NCI, is supporting 30 clinical trials of various chemopreventive agents for premalignant disease in different organ systems that are being monitored by the SEBs of DNA aneuploidy, abnormally high proliferative index, abnormal nuclear morphometry (aberrant size, shape, texture, and pleomorphism) and nucleolar number, position in the nucleus, and morphometry. Evidence from the literature permits the expectation that in premalignant breast disease these SEBs, when measured by computer-assisted quantitative image analysis, will accurately predict the risk of recurrence of breast DCIS after lumpectomy.

Laminin immunohistochemistry: a simple method to visualize and quantitate vascular structures in the mammalian brain

Immunohistochemistry using antiserum against the basement membrane glycoprotein laminin, was shown to be an excellent marker for brain blood vessels. Throughout the brain of mice, rats, guinea pigs, monkeys and humans, the basement membrane of the vascular structures were strongly laminin-positive. The neuropil itself was laminin-negative, whereas a positive reaction was observed in the meninges. When the laminin antiserum was preabsorbed with its proper antigen, no specific fluorescence was observed. Using India ink perfusion as a comparative method, it was found that probably all vascular structures were also visualized with laminin immunohistochemistry. Laminin immunofluorescence was found well-suited for computer-assisted quantitative image analysis of brain vascularity. As expected in the periphery, the basement membrane of many other structures except blood vessels such as endoneurium, epithelium and smooth muscle cells were laminin-positive. Although the vascular network was also strongly laminin-immunoreactive, it was difficult to differentiate between blood vessels and non-vascular structures in the periphery as compared to the central nervous system. In conclusion, laminin immunohistochemistry has proven to be a simple, useful and specific method to study vascular structures in the central nervous system and an excellent alternative to more conventional and laborious methods such as perfusion with India ink.