Computer-assisted Morphometric Analysis Research Articles

Computer-assisted morphometric analysis of the relationship between the coronoid and condylar processes of the mandible

: The mandible is the most durable and only movable bone that constitutes the lower one-third of the face.The mandibular condyle and coronoid processes are two anatomical structures located in the superior portion of the ramus of the mandible. Morphometric understanding of these structures is key to the diagnosis of various disorders associated with the temporomandibular region. It also aids in devising a plan for the management of such disorders.: The purpose of this study is to evaluate the anatomical corelation between the condyle and coronoid of the mandible.: This retrospective study was conducted by obtaining 100 high-resolution computed tomography images from departmental archived records between 2018 and 2024. After acquisition, these images were incorporated into MIMICS Materialize software to create stereolithography models, and morphometric analysis was carried out. Out of 100 HRCT data obtained, 87 were male and 13 were female. There is a statistically significant difference in length and angulation of the coronoid and condyle on both sides. The condyle length for males was (4 ± 5.5 mm) higher than the condyle length of females on both sides. However, the coronoid length of males and females varies in a range of 0.5-2mm on both sides of the mandible. Understanding the morphological variation of mandibular condylar and coronoid processes aids in various fields of medicine like forensics, anthropology, and reconstructive maxillofacial surgery. Our study inference will aid surgeons in planning coronoid grafts for condylar reconstruction as well as other maxillofacial reconstructions. Additionally, it helps in forensic odontology to determine a person's age and gender.

Long COVID in Immunocompromised and Immunocompetent Patients: A Clinical, Morphologic, and Virologic Portrait.

Coronavirus disease 2019 (COVID) primarily affects the lung and can lead to chronic/post-COVID syndrome. Some insights about late pulmonary changes occurring in patients recovering from COVID have been published, but the evidence of detailed pathologic changes coming from follow-up care patients with long COVID is limited. To evaluate tissue morphologic and viral features in transbronchial biopsies of long COVID patients (immunocompetent and immunocompromised). This retrospective observational study included 18 patients (9 immunocompetent and 9 immunocompromised) who were consecutively referred to outpatient clinic for post-COVID pneumonia, undergoing transbronchial biopsy. Several histologic changes were analyzed by computer-assisted morphometric analysis. As organizing pneumonia (OP) was consistently detected, fibrosis and inflammation were also evaluated in transbronchial biopsies from 28 control patients with histologic confirmation of OP. Tissue SARS-CoV-2 and the subgenomic transcripts were investigated. Morphologic findings were correlated with clinical and radiologic data. Long COVID patients showed lower inflammation than controls (P < .001) despite a similar fibrotic extension. When considering separately the 2 long COVID groups, the same inflammatory infiltrate extension was found, whereas a higher fibrotic remodeling characterized the immunocompetent subgroup (P = .05). Molecular investigation showed that SARS-CoV-2 was present in tissue samples obtained from 3 long COVID patients. Long COVID patients showed a peculiar OP pattern, with more vascular and fibrotic changes. SARS-CoV-2 RNA, even in replicative status, can be detected in lung biopsies of both immunocompetent and immunocompromised patients. This pilot study is a forerunner of more in-depth lung tissue investigations to gain a better understanding of long COVID pathobiology.

Computer-Assisted Morphometric Comparative Analysis of Argyrophilic Nucleolar Organizer Regions (AgNORs) in Leukoplakia With Dysplasia and Oral Squamous Cell Carcinoma.

Background Oral squamous cell carcinoma (OSCC) and oral leukoplakia (OL) with dysplasia are closely linked conditions in the oral cavity, with the latter often indicating precancerous changes, underscoring the urgency of early detection and intervention. Histopathological confirmation is crucial for accurate diagnosis. The nucleolar organizer region (NOR), specifically analyzed through silver-staining (argyrophilic NORs), provides insights into nuclear changes associated with the lesion. Computer-assisted morphometric analysis enhances precision and objectivity in evaluating AgNOR-related parameters. Aim To conduct a computer-assisted morphometric comparison of AgNORs using various NOR-related parameters in cases of OSCC and leukoplakia with dysplasia and to evaluate their diagnostic significance. Materials and methods A computer-assisted morphometric analysis was conducted using various NOR-related parameters, such as nuclear profile area, single AgNOR profile area per nucleus, total AgNOR profile area per nucleus, and number of AgNOR profiles per nucleus on a total sample of 90 specimens, which includes leukoplakia with dysplasia (30), OSCC (30), and a control group, including 30 samples of normal oral mucosa. A comparison was conducted on the morphometric values between the groups under investigation. Tukey's multiple comparison tests and ANOVA were used to analyze the data and determine the differences between the groups. Results The present investigation revealed a significant difference in all four AgNOR-related parameters between leukoplakia and OSCC in comparison to the control group (normal oral mucosa). Comparing OL (41.78 ± 0.46) and OSCC (62.78 ± 0.47) to the control group (35.93 ± 0.99), the mean value of nuclear profile area (A Nuc) was significantly greater. In comparison to the control group (3.40 ± 0.09), the mean value of a single AgNOR profile area per nucleus (A NOR) was found to be relatively lower in both research groups, OL (2.00 ± 0.02) and OSCC (1.39 ± 0.01). The total AgNOR profile area per nucleus (TA NOR) had a mean value of 10.61 ± 0.69 in OL and 12.05 ± 0.28 in OSCC, respectively, compared to 7.82 ± 0.38 in the control group. The study found that there was more number of profiles of AgNORs per nucleus (n NOR)in the study groups of OL (5.30 ± 0.29) and OSCC (8.69 ± 0.19) than in thecontrol group (2.32 ± 0.11). Conclusion The parameters linked to the NOR are biologically informative and easy to check regularly in a pathology lab. Additionally, AgNORs give us important information that enables us to study the range of nuclear changes in malignant and potentially malignant lesions.

Enhancing structural plasticity of PC12 neurons during differentiation and neurite regeneration with a catalytically inactive mutant version of the zRICH protein

BackgroundStudies of the molecular mechanisms of nerve regeneration have led to the discovery of several proteins that are induced during successful nerve regeneration. RICH proteins were identified as proteins induced during the regeneration of the optic nerve of teleost fish. These proteins are 2’,3’-cyclic nucleotide, 3’-phosphodiesterases that can bind to cellular membranes through a carboxy-terminal membrane localization domain. They interact with the tubulin cytoskeleton and are able to enhance neuronal structural plasticity by promoting the formation of neurite branches.ResultsPC12 stable transfectant cells expressing a fusion protein combining a red fluorescent protein with a catalytically inactive mutant version of zebrafish RICH protein were generated. These cells were used as a model to analyze effects of the protein on neuritogenesis. Differentiation experiments showed a 2.9 fold increase in formation of secondary neurites and a 2.4 fold increase in branching points. A 2.2 fold increase in formation of secondary neurites was observed in neurite regeneration assays.ConclusionsThe use of a fluorescent fusion protein facilitated detection of expression levels. Two computer-assisted morphometric analysis methods indicated that the catalytically inactive RICH protein induced the formation of branching points and secondary neurites both during differentiation and neurite regeneration. A procedure based on analysis of random field images provided comparable results to classic neurite tracing methods.Graphical

MP41-13 QUANTITATIVE ASSESSMENT OF MESENCHYMAL DIFFERENTIATION FOR METASTATIC RISK PREDICTION OF CLEAR CELL RENAL CARCINOMA (CCRCC)

You have accessJournal of UrologyCME1 Apr 2023MP41-13 QUANTITATIVE ASSESSMENT OF MESENCHYMAL DIFFERENTIATION FOR METASTATIC RISK PREDICTION OF CLEAR CELL RENAL CARCINOMA (CCRCC) Daniel Vilchez, Bodong Zhang, Alejandro Sanchez, Deepika Sirohi, Tolga Tasdizen, and Beatrice Knudsen Daniel VilchezDaniel Vilchez More articles by this author , Bodong ZhangBodong Zhang More articles by this author , Alejandro SanchezAlejandro Sanchez More articles by this author , Deepika SirohiDeepika Sirohi More articles by this author , Tolga TasdizenTolga Tasdizen More articles by this author , and Beatrice KnudsenBeatrice Knudsen More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003279.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Epithelial-to-mesenchymal transition (EMT) results in poor prognosis sarcomatoid and rhabdoid histologic growth patterns (HGP) of ccRCC. However, only ∼15% of metastatic cases reveal areas of sarcomatoid/rhabdoid differentiation in the primary tumor. We questioned whether a computer can learn salient features of EMT that are associated with risk of metastatic progression across all HGPs of ccRCC. METHODS: The project uses 410 digital H&E slides of ccRCC. We incorporated a novel co-training approach into a machine learning model (MLM) and trained the model to distinguish epithelial (nested) and mesenchymal (sarcomatoid, rhabdoid) HGPs in a Utah cohort. The trained MLM is applied to the TCGA KIRC cohort (n=360) with annotated regions of nested, tubulo-papillary, low grade-spindle, sarcomatoid and rhabdoid HGPs for which the hazard ration of metastatic progression was published. For each case, the MLM outputs a mesenchymal-differentiation (DP-MD) score on a scale of 0 to 1 where closer to 1 is higher likelihood of EMT. RESULTS: A unique machine learning / AI model was developed to quantify sarcomatoid differentiation in ccRCC with no obvious histologic signs of mesenchymal morphology. H&E stains were used as separate inputs of a contrastive co-training model (H/E-CCTM) to force the learning of mesenchymal features. The inclusion of a semi-supervised learning component improved the accuracy of predicting mesenchymal differentiation by >4% over other state-of-the-art co-training algorithms and performed equally well when trained on 10% versus 100% of labeled cancer data. Sarcomatoid, low grade spindle and rhabdoid cases received average DP-MD scores of 0.91, 0.97 and 0.83, respectively in a held-out test set, while cases with nested morphology, including expansile, and high nuclear to cytoplasmic ratio averaged DP-MD scores of 0.08. Tubulo-papillary HGPs without obvious mesenchymal features obtained an average DP-MD score of 0.49. CONCLUSIONS: Computer-assisted morphometric analysis can be used to quantify mesenchymal differentiation and has the potential to improve the prediction of metastatic risk in ccRCC and the selection of patients for adjuvant treatment after nephrectomy. Source of Funding: Huntsman Cancer Foundation, NIH P30CA042014 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e560 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniel Vilchez More articles by this author Bodong Zhang More articles by this author Alejandro Sanchez More articles by this author Deepika Sirohi More articles by this author Tolga Tasdizen More articles by this author Beatrice Knudsen More articles by this author Expand All Advertisement PDF downloadLoading ...

Expression Level and Biomarker Potential of Hemoxygenase-1 in Pancreatic Cancer

<h3>Purpose/Objective(s)</h3> In pancreatic ductal adenocarcinoma (PDAC), hypoxia is key mediator of resistance to chemo-radiotherapy and a prominent feature of PDAC tumor microenvironment (TME). Heme oxygenase-1 (HMOX-1) is an Nrf2-regulated gene that plays a critical role in modulation of oxidative stress and ROS production. Previous research suggested that HMOX-1 is expressed in the hypoxic PDAC TME and is induced upon exposure to chemotherapy. Utilizing a tumor microarray (TMA) of PDAC patients treated at our institution, we sought to determine baseline expression of HMOX-1 in the tumor and the TME and whether it could be a biomarker of resistance to neoadjuvant treatment (NAT). <h3>Materials/Methods</h3> Four TMAs containing 80 cores (of normal and cancerous pancreatic tissue) on each slide were stained with HMOX-1 antibody. Slides were manually evaluated and computer-assisted morphometric analysis of digital images was performed using the Aperio Image Analysis software™. Immunohistochemistry (IHC) staining was manually scored as follows: Tumor cells staining, Normal and hyperplasia cell staining, TME staining, Intensity of IHC stain (negative = 0, borderline = +/-, minimal = 1, moderate = 2, or severe = 3); Localization of stain and its distribution (homogenous, heterogeneous, focal, multifocal, and/or variable). The Positive Pixel Count algorithm was used to quantify the amount of stain present in a scanned slide image and derive the H-score which represents a combination of number weak positives, moderate positives, and strong positives/Number of Total positive/ pixels on each core. <h3>Results</h3> Through the Oncology Research Information Exchange Network (ORIEN) program, we developed 6 TMAs of PDAC patients that were treated at our institution between 2013-2020. Four out of 6 available PDAC TMAs were evaluated for expression of HMOX-1 equaling to 320 cores representing 101 patients. 35% of those patients received NAT. Seven out of 14 patients who received RT as part of their NAT were treated with SBRT. A total of 175 eligible cores were included in the final analysis. Overall, there was minimal cytoplasmic cell staining in pancreatic cancer cells. Only 5 out of 175 eligible cores were considered true positives. The 5 positive cores represented 5 different patients. None of these patients received NAT. One true positive underwent whole genome sequencing and was found to have a BRIP1 (BRCA-1 interacting protein) germline mutation. There was no staining in the TME and mild inflammatory cell staining (macrophages and subset of lymphocytes). <h3>Conclusion</h3> We showed that HMOX-1 is not overexpressed at baseline in pancreatic cancer tissue, limiting its use as a predictive marker for resistance to NAT. However, given its inducible nature, it could be primarily expressed upon exposure to these therapies. Future research is needed to investigate mechanism of HMOX-1 induction after treatment. Our future goal is to utilize this clinically rich resource to investigate other relevant proteins that may predict response to therapy.

JQ-1 ameliorates schistosomiasis liver fibrosis by suppressing JAK2 and STAT3 activation

Schistosomiasis is a serious parasitic infection caused by Schistosoma. The parasite deposits eggs in the host liver, causing inflammation that activates hepatic stellate cells (HSCs), which leads to liver fibrosis. Currently, there is no effective therapy for liver fibrosis; thus, treatments are urgently needed. Therefore, in the present study, mice infected with Schistosoma japonicum were treated with JQ-1, a small-molecule bromodomain inhibitor with reliable anti-tumor and anti-inflammatory activities. The fibrotic area of the liver measured by computer-assisted morphometric analysis and the expression levels of the cytoskeletal protein alpha smooth muscle actin (α-SMA) and of collagen assessed by quantitative PCR, Western blot and immunohistochemistry were significantly decreased in the liver following JQ-1 treatment compared with vehicle-treated controls. Total RNA was extracted from the liver of JQ-1–treated Schistosoma-infected mice for RNA-sequencing analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that JQ-1 affected biological processes and the expression of cellular components known to play key roles in the transdifferentiation of HSCs to myofibroblasts. In vitro treatment with JQ-1 of JS-1 cells, a mouse HSC line, indicated that JQ-1 significantly inhibited JS-1 proliferation but had no effect on JS-1 activity, senescence, or apoptosis. Western blot results showed that JQ-1 inhibited the expression levels of phosphorylated JAK2 and phosphorylated STAT3 without altering expression levels of these non-phosphorylated proteins. Taken together, these findings suggested that JQ-1 treatment ameliorated S. japonicum egg–induced liver fibrosis, at least in part, by suppressing HSC activation and proliferation through the inhibition of JAK2/STAT3 signaling. These results lay a foundation for the development of novel approaches to treat and control liver fibrosis caused by S. japonicum.

Effects of maternal diazinon exposure on frontal cerebral cortical development in mouse embryo

Background Diazinon including organophosphate (OP) that is widely used in agriculture and animal husbandry industry and the risk of human infection with the toxin and their toxicity. Methods Pregnant balb/c mice (30–35 g) were randomly divided into five groups of five: the control group (no intervention), two sham groups (emulsifier 0.52, and 5.2 liters/volume). From the seventh to the eighteenth day of pregnancy, two experimental groups received diazinon inhaled 1.3 (EXP1) and 13 liters/volume (EXP2) for 40 min every other day, respectively. On the 18th day of pregnancy, the animals were killed and their embryos were removed to appraisal the growth of fetus and development of the frontal cortex. A computer-assisted morphometric quantitative images analysis were performed on the frontal cerebral cortex (FCC) including cortical plate (CP), intermediate zone (IZ) and matrix (proliferative) zone (MZ) of the mouse embryos. Findings The average of crown-rump length and weight of the embryos in the experimental groups were increased without any significant difference. The mean fetal FCC thickness in the EXP2 group was significantly reduced compared to the control group, CP thickness was remarkably increased in fetuses exposed to diazinon. Comparing the mean thickness of MZ and IZ in EXP groups with the sham and control groups indicated a significant decrease. The positive K-67 cells in the FCC of the EXP2 group were significantly reduced. Discussion Exposing diazinon during pregnancy can reduce brain development and would be neurotoxic to the developing brain and can lead to behavioral changes in the offspring.

An in situ and morphometric study of maize (Zea mays L.) cob rondel phytoliths from Southwestern North American landraces

We present the first comprehensive computer-assisted morphometric analysis of microscopic rondel11As per the International Code for Phytolith Nomenclature 2.0 the names of recognized phytolith morphotypes are written in small caps in this report (Neumann et al., 2019). phytoliths (plant opal microfossils) produced in the cobs of 24 historic Southwestern North American landraces of maize (Zea mays L.) after all were grown in a well-documented agronomic field study. We also present an in situ study of the location of rondel phytolith production within the maize cob and provide a detailed review of previous maize phytolith studies. We found that glumes contained abundant rondel phytoliths throughout the tissue; however, lemma/palea tissue contained no phytoliths. In contrast, cupule tissue had some areas with abundant phytoliths, some with fewer scattered phytoliths, and vast areas that contained no rondel phytoliths. The rondel-rich areas appear to be where the glumes had once attached to the cupule and may be remnants of glume tissue adhering to the cupule. From the morphometric study, we found there were significant differences in the size morphometries of glume rondels depending on their cob location (top, middle, base) but no significant differences in shape morphometries. Using shape morphometries, we could not discriminate reliably among maize cob rondel phytoliths produced by the diverse landraces considered. The inclusion of morphometrics from areas in addition to or in combination with the outer periclinal surface may allow for some discrimination of maize landraces and is an avenue that should be explored further. Although our approach was not successful at identifying differences between essentially modern landraces, there may be significant rondel phytolith morphometric differences between wild, progenitor, and domesticated Zea.

Morphometric Analysis of Suprabasal Cell Layer in Oral Epithelial Dysplasia: A Computer-assisted Microscopic Study.

ABSTRACTBackground:Oral leukoplakia is the most common precancerous lesion. Various grading systems based on histological findings have been proposed for dysplasia. Recently, computer-assisted morphometric analysis has been established to reduce the interobserver and intraobserver variability during the histological grading of epithelial dysplasia. This study was undertaken to establish the morphometric changes in the suprabasal cell layer of different grades of oral epithelial dysplasia.Materials and Methods:Forty paraffin-embedded tissue sections (10 normal mucosa, 10 mild dysplasia, 10 moderate dysplasia, and 10 severe dysplasia cases) were stained using hematoxylin and eosin stain, and analyzed for cellular and nuclear morphometry using binocular microscope.Results:Our results showed that values of nuclear area, nuclear perimeter, nuclear volume density, and nuclear/cytoplasmic (N/C) ratio were increased gradually in dysplasia compared to control groups and the values were statistically significant (P = 0.001). Nuclear diameter and cellular area were increased in dysplasia when compared to control group (P = 0.001). Mild and moderate dysplasia showed decreased value of nuclear form factor compared to control group, whereas severe dysplasia showed highest value. A fair correlation was found when comparing histological grading and grouping based on nuclear area, nuclear perimeter, N/C ratio, and nuclear volume density.Conclusion:Nuclear features reflect cell behavior, and its morphometric analysis can be considered as a reliable tool for differentiating various grades of epithelial dysplasia.

Histological Classification of Thymoma

Background: Many thymoma classifications have been followed and have been updated by newer or alternative schemes. Many classifications were based on the morphology and histogenesis of normal thymus as the backbone, while other classifications have followed a more simplified scheme, whereby thymomas were grouped based on biological behavior. The WHO classification is currently the advocated one, which is based on “organotypical” features (i.e. histological characteristics mimicking those observed in the normal thymus) including cytoarchitecture (encapsulation and a “lobular architecture”) and the cellular composition, mostly the nuclear morphology is generally appreciated.&#x0D; Objectives: This study aims to re-classify thymomas by establishing certain morphometric parameters to evaluate the epithelial cells nuclei. An appraisal of thymoma classification as cortical/ lymphocytic/ type B1 and B2 or medullary/ spindle cells/ type A will be attempted as objective re-evaluation of thymoma.&#x0D; Patients: This study is a retrospective evaluation of 50 cases of thymoma, 20 cases of thymic hyperplasia and 10 cases of normal thymus (control group). Using a 5 µm formalin-fixed paraffin embeded tissue sections, stained with hematoxylin-eosin stain, these cases were previously classified histologically into lymphocytic (6 cases), lymphoepithelial (mixed) (28 cases) and epithelial (16 cases), including 2 cases of spindle cell thymoma.&#x0D; Methods: Computer-assisted morphometry was performed for 80 cases. This involves digitization of the histopathological features and application of morphometric analysis through software. The morphometric parameters used are nuclear area, maximum nuclear diameter and Form factor of epithelial cells nuclei. Ten normal thymus glands from the archived cases were also evaluated as a control groups.&#x0D; Results: The results showed that epithelial thymomas possess significantly different nuclear areas from that of a normal thymus, the maximum nuclear diameter (D-Max) follows the same pattern and adds no further outcomes. The numerical morphometric analysis showed no significant differences between lymphocytic predominant thymoma and those classified as cortical thymoma (Type B2). Thus it does not support such a re-classification. Form factor is an indication of pleomorphism, but it should be cautiously used when spindle cells are present since it may give a false indication of pleomorphism.&#x0D; Conclusion: Computer-Assisted morphometric analysis provides an objective, reproducible and comparable results for thymoma histological classification.&#x0D; Keywords: Thymoma, Histological classification, Grading, Morphometry, Computer-assistedmorphometry.

Increased mitochondrial fission of glomerular podocytes in diabetic nephropathy.

AimsPrevious studies showed that abnormal mitochondrial structure and function were involved in the pathological process of diabetic nephropathy (DN). The dynamic mitochondrial processes, including fusion and fission, maintain the mass and quantity of mitochondria. Podocyte injury is a critical factor in the development and progression of DN. The present study evaluated the mitochondrial fission of podocytes in patients with DN.MethodsWe recruited 31 patients with biopsy-confirmed DN. A quantitative analysis of the mitochondrial morphology was conducted with electron microscopy using a computer-assisted morphometric analysis application to calculate the aspect ratio values. Immunofluorescence assays were used to evaluate protein colocalization in the glomeruli of patients.ResultsThe urine protein level was significantly increased in DN patients compared to non-DN patients (P < 0.001), and the mitochondria in the podocytes from DN patients were more fragmentated than those from patients without DN. The mitochondrial aspect ratio values were negatively correlated with the proteinuria levels (r = −0.574, P = 0.01), and multiple regression analysis verified that the mitochondrial aspect ratio was significantly and independently associated with the urine protein level (β = −0.519, P = 0.007). In addition, Drp1, a mitochondrial fission factor, preferentially combines with AKAP1, which is located in the mitochondrial membrane.ConclusionsIn the podocytes of DN patients, mitochondrial fragmentation was increased, and mitochondrial aspect ratio values were correlated with the proteinuria levels. The AKAP1-Drp1 pathway may contribute to mitochondrial fission in the pathogenesis of DN.

Survival prediction in patients with cutaneous melanoma by tumour lymphangiogenesis

ABSTRACT Objectives: Melanoma induces lymphangiogenesis by secreting lymphangiogenic growth factors. The aim of this study was to examine the role of tumour lymphangiogenesis in survival of patients with cutaneous melanoma. Methods: Immunostaining of one hundred melanoma specimens was done with lymphatic-specific antibody D2-40. The quantification of tumour lymphangiogenesis – lymphatic vessel density (LVD) and lymphatic vessel area (LVA) – was calculated by computer-assisted morphometric analysis. Results: High intratumoural LVD, high peritumoural LVD, male gender, greater tumour thickness and Clark level IV/V were significantly associated with shorter disease-free survival (p= 0.001, p= 0.004, p= 0.004, p= 0.000 and p= 0.008, respectively) and melanoma-specific survival (p= 0.002, p= 0.002, p= 0.001, p= 0.000 and p= 0.017, respectively), while the trunk melanoma site was significantly associated only with shorter disease-free survival (p= 0.033). No significant association of LVA with survival was found. At multivariate analysis, peritumoural LVD [hazard ratio (HR) = 2.143, 95% confidence interval (CI) 1.097–4.189, p= 0.026)] and melanoma thickness (HR = 1.276, 95%CI 1.106–1.473, p= 0.001) were independent predictors of disease-free survival, while intratumoural LVD (HR = 3.446, 95%CI 1.465–8.109, p= 0.005), peritumoural LVD (HR = 2.742, 95%CI 1.313–5.725, p= 0.007) and gender (HR = 2.880, 95%CI 1.304–6.362, p= 0.009) were independent predictors of melanoma-specific survival. Conclusion: Тhis study shows that LVD enables better prediction of survival than melanoma thickness and other clinical-pathological parameters. Intratumoural LVD is the most significant predictor of melanoma-specific survival, while only peritumoural LVD has a significant impact on both, a disease-free survival and a melanoma-specific survival.

Role of Nuclear Morphometry in Breast Cancer and its Correlation with Cytomorphological Grading of Breast Cancer: A Study of 64 Cases.

Background:Fine needle aspiration cytology (FNAC) is a simple, rapid, inexpensive, and reliable method of diagnosis of breast mass. Cytoprognostic grading in breast cancers is important to identify high-grade tumors. Computer-assisted image morphometric analysis has been developed to quantitate as well as standardize various grading systems.Aims:To apply nuclear morphometry on cytological aspirates of breast cancer and evaluate its correlation with cytomorphological grading with derivation of suitable cutoff values between various grades.Settings and Designs:Descriptive cross-sectional hospital-based study.Materials and Methods:This study included 64 breast cancer cases (29 of grade 1, 22 of grade 2, and 13 of grade 3). Image analysis was performed on Papanicolaou stained FNAC slides by NIS –Elements Advanced Research software (Ver 4.00). Nuclear morphometric parameters analyzed included 5 nuclear size, 2 shape, 4 texture, and 2 density parameters.Results:Nuclear size parameters showed an increase in values with increasing cytological grades of carcinoma. Nuclear shape parameters were not found to be significantly different between the three grades. Among nuclear texture parameters, sum intensity, and sum brightness were found to be different between the three grades.Conclusion:Nuclear morphometry can be applied to augment the cytology grading of breast cancer and thus help in classifying patients into low and high-risk groups.

Lymphatic vessel density and VEGF-C expression as independent predictors of melanoma metastases

In many patients, the clinical behaviour of cutaneous melanoma is very difficult to predict by traditional histologic and clinical parameters. This study aimed to examine the role of quantitative parameters of tumour lymphangiogenesis and vascular endothelial growth factor (VEGF)-C in predicting metastatic risk in patients with cutaneous melanoma. One hundred melanoma specimens were stained with lymphatic-specific antibody D2-40 and with anti-VEGF-C antibody. Quantitative parameters of lymphangiogenesis-lymphatic vessel density (LVD) and lymphatic vessel area (LVA)-were determined by computer-assisted morphometric analysis. Moderate or strong staining was assessed as a positive expression of VEGF-C in tumour cells. Univariate analysis revealed that intratumoural LVD, peritumoural LVD, VEGF-C expression in tumour cells, melanoma thickness, Clark level, ulceration, gender and histologic type were significant predictors of lymph node metastasis (p=0.000, p=0.000, p=0.000, p=0.000, p=0.005, p=0.005, p=0.011 and p=0.027, respectively). No significant association of intratumoural and peritumoural LVA with metastases was found. In multivariate analysis, independent predictors of metastatic risks were melanoma thickness [odds ratio OR=1.655, 95% confidence interval (CI) 1.102-2.484, p=0.015], intratumoural LVD (OR=1.086, 95% CI 1.027-1.148, p=0.004), peritumoural LVD (OR=1.050, 95% CI 1.008-1.094, p=0.020) and a positive VEGF-C expression in tumour cells (OR=20.337, 95% CI 2.579-160.350, p=0.004). This study identified intratumoural and peritumoural LVD and the VEGF-C expression in tumour cells as more significant predictors of metastatic risk than melanoma thickness, ulceration and other clinical-pathological parameters.

Tumor vascularization and clinicopathologic parameters as prognostic factors in merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine tumor of the skin with an increasing incidence. The clinical course is variable and reliable prognostic factors are scarce. Tumor angiogenesis has been shown to have prognostic impact in different types of cancer. The aim of our study was to determine potential prognostic factors, including tumor vascularization, for clinical outcome of MCC. The medical records of 46 patients with MCC diagnosed between 1997 and 2010 were analyzed retrospectively. Tissue samples were immune-stained for the lymphatic endothelial vessel marker podoplanin/D2-40 and the panvascular marker CD31. These immunostained sections were analyzed using computer-assisted morphometric image analyses. Aside from the parameters of tumor vascularization, clinicopathologic features were investigated, and progression-free survival (PFS) and tumor-specific survival (TSS) were assessed. Univariate and multivariate analyses were performed to determine prognostic factors. Male sex of the MCC patients and a high cross-sectional whole vessel area (WVA) in relation to the entire tumor area as determined on CD31-stained tumor sections were found to be negative prognostic factors for PFS in a univariate and multivariate regression analysis. Ulceration of the primary tumor was significantly associated with both impaired PFS and TSS. Our results indicate a high prognostic impact of tumor vascularization on the clinical outcome of MCC patients. Male sex and ulceration of the primary MCC were identified as independent unfavorable prognostic markers for the clinical outcome. As an outlook, MCC patients with increased angiogenesis might be identified and subjected to a targeted anti-angiogenic treatment.

Computer-assisted morphometric image analysis of cells of normal oral epithelium and oral squamous cell carcinoma.

Context:Oral squamous cell carcinoma (OSCC) is a malignant neoplasm of epithelial tissue origin. OSCC is traditionally graded into well, moderate and poorly differentiated. The assessment of cellular and nuclear features is very important in the histological grading of OSCC.Aims:To establish morphometric study as an important prognostic indicator of OSCC.Settings and Design:The present study is undertaken for computer-assisted morphometric evaluation of the following parameters: changes in cell perimeter, nuclear perimeter (NP), cell area (CA), nuclear area (NA) and nuclear-cytoplasmic ratio (N:C) in twenty cases of OSCC and ten cases of normal oral mucosa.Subjects and Methods:The hematoxylin- and eosin-stained sections were observed under microscope for dysplastic features. For morphometric analysis, images were captured with a 2MP camera attached to a NLCD 307 microscope (Lawrence and Mayo India Pvt., Ltd.,) with a ×40 objective used for morphometric analysis. The images were classified, transferred and stored in the computer for image analysis.Statistical Analysis Used:One-way analysis of variance is used for comparing the parameter for multiple groups followed by Mann-Whitney U-test for pair-wise comparisons.Results:Highly significant difference was seen between cases and controls with respect to CA, NP and NA. Highly significant difference is seen in N:C ratio between the means of cases and control groups.Conclusions:Techniques of image analysis offer an opportunity to quantify the nuclear and cell changes associated with malignancy and provide an objective basis for grading dysplasia and tumors.

Prognostic value of the area and density of lymphatic vessels in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (SCC) is known for its capacity to metastasize via lymphatic vessels. In recent studies, the level of lymphangiogenesis has been reported as a potential prognostic factor for several skin tumors. The aim of this study was to quantify lymphangiogenesis in SCC using either computer-assisted image analysis or the Chalkley count technique. Vascular parameters were evaluated and compared with respect to their predictive power for tumor metastasis. In this case-control study, clinical and histological data of 15 metastatic and 15 nonmetastatic SCC patients were retrospectively analyzed. SCC samples were immunostained for the lymphatic endothelial marker D2-40 and the panvascular marker CD31, and analyzed using computer-assisted morphometric image analyses within hot spots as well as the digitalized Chalkley counting method. Lymphatic vessel density, relative lymphatic vessel area, and lymphatic Chalkley count were significantly elevated in metastatic SCC. Tumor thickness was significantly higher in metastatic SCC, and had the highest predictive power for metastatic disease. Tumor thickness was a significant predictor of lymphangiogenic parameters. Lymphangiogenesis is elevated in metastatic SCC but its extent is influenced by tumor thickness. Tumor thickness remains the most reliable predictive factor for metastatic disease.

Abstract 4940: Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas

Abstract Pancreatic cancer is the 4th most common cause of cancer deaths in this country. Close to 50,000 deaths occur every year. This is due to the fact that most patients are not diagnosed until presentation at a stage IV. In a recent study, it is estimated that more than 10 years go by between the first genetic mutation and the formation of invasive cancer. More mutations occur in metastatic lesions after an additional 6 years. Both SNAIL and TWIST have gained more attention as biomarkers because that contribute to E-cadherin dysregulation that allows the tumor cells to invade. In this study we evaluated 17 cases of pancreatic ductal adenocarcinoma using twist-1 antibody to evaluate tumor cell expression in these cases. All paraffin blocks were retrieved from the IU pathology department files via an IRB approved protocol. The unstained slides were stained with primary antibody Twist-1 using the DAKO Flex system. Control sections were treated with an isotype control using the same concentration as primary antibody to verify the staining specificity. The Aperio whole slide system was used to image the slides. Computer-assisted morphometric analysis of digital images was carried out using the Aperio Image scope software using the positive pixel algorithm. The algorithm was used to quantify the Twist-1 positivity of normal pancreas, and pancreatic ductal adenocarcinoma. This algorithm is the only FDA approved algorithm for human clinical trials. The immunostaining of Twist-1 was found only in the ductal epithelial carcinoma cells in the pancreas. There was no staining in the stroma in any of the normal pancreas and ductal pancreatic carcinoma cases. We analyzed 5 normal pancreata, and 12 pancreatic ductal adenocarcinomas. The data from the image analysis was 3.38% expression in normal pancreata and 11.44% expression in the pancreatic ductal adenocarcinoma. In summary, the image analysis data demonstrates the overexpression of Twist-1 in stage IV pancreatic adenocarcinomas compared to the normal pancreas. This suggests that this marker may be a predictor of tumor invasiveness and metastasis. Citation Format: Max Jacobsen, George Sandusky, Kavita Shah. Use of whole slide digital image analysis for determination of expression of Twist-1 in normal panceata and stage IV pancreatic ductal adenocarcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4940.

Abstract 426: Identifying a predictive biomarker for chemotherapy (taxane) in patients with metastatic castration resistance prostate cancer

Abstract In 2015, American Cancer Society predicts that 220,800 men will be diagnosed with prostate cancer (PC). Patients diagnosed with metastatic PC are first treated with androgen deprivation therapies (ADT) in order to lower testosterone to castration levels. Unfortunately, it is only a matter of time before becoming metastatic castration resistant prostate cancer (mCRPC). Subsequent treatments include combined androgen deprivation therapies such as (abiraterone, enzalutamide), immunotherapies, investigational drugs, and/or chemotherapy (docetaxel and cabazitaxel). Individualizing therapy for prostate cancer patients is an overarching goal of research. Currently, no valid practical biomarkers exist that can predict resistance to the taxane in PC. Identifying a marker that predicts taxane sensitivity would delineate patients who would benefit from taxane regimens from those who would suffer from added side-effects without any improved response. Previous studies have confirmed TLE3 is associated with a response to taxane therapy in breast and ovarian cancers. Preclinical studies have also shown that TLR4, RAGE, and HMGB1 confers chemo resistance to docetaxel on PC-3 human PC cells. After obtaining IRB approval (protocol IUCRO-0499), 8 metastatic castration resistant prostate cancer samples and 6 control samples were pulled for this study after scrutinous review of the H&amp;E slides from clinical cases. This study then consisted of investigating the proteins TLE3, TLR4, RAGE, and HMBG1. Immunostaining was performed using the automated immunostainer, Dako FLEX System, and the slides were scanned into the whole slide Aperio Scanscope CS digital imaging system. Computer-assisted morphometric analysis of digital images was then performed using the Aperio ImageScope software. Review of the TLE3 and TLR4 expression in metastatic castration resistance prostate cancer samples showed a difference between the expressions of these two proteins. TLR4 positivity was found to be 43.88% higher than the expression of TLE3. This is suggestive of resistance to docetaxel treatments. Review of the RAGE and HMGB1 expression in metastatic castration resistance prostate cancer samples compared to normal prostate epithelium showed a 32.9% increase in RAGE and 36.6% increase in HMGB1 expression. The findings of this study are suggestive that there is a possible correlation between the overexpression of these protein biomarkers and the patient's response to a specific drug treatment. Citation Format: Greg A. Rothchild, George Sandusky, Constance J. Temm, Costantine Albany. Identifying a predictive biomarker for chemotherapy (taxane) in patients with metastatic castration resistance prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 426.