Immunohistochemical Preparations Research Articles

DNA content and clinicopathological features aid in distinguishing ameloblastic carcinoma from ameloblastoma.

Ameloblastoma and ameloblastic carcinoma are epithelial odontogenic tumors that can be morphologically similar. In the present study, we evaluated the DNA content and Ki-67 index in the two tumors. The paraffin blocks of the tumors were selected to obtain sections for the immunohistochemical reactions and preparation of the cell suspension for acquisition in a flow cytometer. The Random Forest package of the R software was used to verify the contribution of each variable to classify lesions into ameloblastoma or ameloblastic carcinoma. Thirty-two ameloblastoma and five ameloblastic carcinoma were included in the study. In our sample, we did not find statistically significant differences in Ki-67 labeling rates. A higher fraction of cells in 2c (G1) was correlated with the diagnosis of ameloblastoma, whereas higher rates of 5c-exceeding rate (5cER) were correlated with ameloblastic carcinoma. The Random Forest model highlighted histopathological findings and parameters of DNA ploidy study as important features for distinguishing ameloblastoma from ameloblastic carcinoma. Our findings suggest that the parameters of the DNA ploidy study can be ancillary tools in the classification of ameloblastoma and ameloblastic carcinoma.

P18.02.B RELATIONSHIP BETWEEN PROGESTERONE RECEPTOR EXPRESSION WITH HISTOPATHOLOGICAL GRADING IN MENINGIOMA

Abstract BACKGROUND Meningioma is a group of mostly benign, slow-growing neoplasms that most likely derive from meningothelial cells of the arachnoid layer of the brain and spinal medulla. Meningiomas are found in one-third of all tumors in the central nervous system. Meningioma is three times more prevalent in women than in males, it is frequently related to hormones in women, particularly progesterone, which is considered to have the utmost impact on meningioma. Objective: this study aims at discovering the relationship between progesterone receptor expression and meningioma histopathological grading in Indonesia, especially at Saiful Anwar Hospital Malang. Method: This study employed a cross-sectional analytical observational research design. The study sample was a paraffin block of meningioma cases at The Anatomical Pathology laboratory of Saiful Anwar Hospital and The Medical Faculty of Brawijaya University, obtained from 2017 to 2020. The data were collected through histopathological preparations, immunohistochemical preparations, and immunohistochemical evaluation. The data analysis was performed by using SPSS version 20. RESULTS Most meningioma cases at Dr. Saiful Anwar Malang Hospital are women between 41 and 50 years. Then, it is located in the frontal part and acquired a grade of histopathology I and has a progesterone receptor expression score of 4. The chi-square test showed no significant association between progesterone receptor expression and histopathological grading of meningioma. At the same time, partial logistic regression tests showed no significant association between sex, age, and location with meningioma histopathological grading. In addition, multinomial logistic regression tests showed that the sex, age, and location did not significantly increase or decrease the risk (odds ratio) to the grade of meningioma histopathology. CONCLUSION This study showed no significant association between progesterone receptor expression and meningioma histopathological grading and no association between the sex, age, and location of lesions with meningioma histopathological grading and no significant increased risk of histopathological grading due to gender, age, or lesion location.

Prognostic value of CD4<sup>+</sup> in patients with Т1–2N0M0 breast cancer

Aim. To evaluate the prognostic value of CD4+ in patients with T1–2N0M0 breast cancer.Materials and methods. We performed a quantitative assessment of CD4+ T-lymphocytes in tumor stromal cells and determined the prognostic parameter using the method developed by us (patent No. RU2697709C1 dated 19.08.2019) in 394 patients diagnosed with infiltrative breast cancer. Survival of patients in the analyzed group was assessed over a period of 10 years.Archival material from paraffin blocks of the «target group» was used in the work for molecular genetic profiling techniques (T1–2N0M0, 1118 patients, 2000–2009). A histological preparation stained with hematoxylin and eosin was scanned, and the central and peripheral zones of the tumor were isolated. To identify CD4+ T-lymphocytes, the immunohistochemical study of preparations obtained with rabbit anti-CD4 monoclonal antibodies was performed. After rescanning histological and immunohistochemical preparations, combining different immunohistochemical stains, the cell density in the tumor and stromal components was calculated in preparations stained with hematoxylin and eosin and CD4. For further calculations, we used the data obtained as a result of the number of stromal cells reduced to 1 mm2 (according to the preparation stained with hematoxylin and eosin), the so-called cell density per calculated number of CD4+-stained cells (cell area × proportion of CD4+ cells in percentages). The resulting ratio is used to predict the outcome of breast cancer: if its value is ≤ 50 %, the prognosis is assessed as favorable (in which the patient’s survival is 10 years or more).Results. In 296 (75 %) of 394 observed patients, the proportion of CD4+ T-lymphocytes in tumor stromal cells was ≤ 50 %. Over 10 years, the overall survival of patients in this group was 93 %, which is considered a statistically favorable group for breast cancer (stages T1–2, N0). In 98 (25 %) of 394 observed patients, the proportion of CD4+ T-lymphocytes in tumor stromal cells was ˃50 %. Over 10 years, the overall survival of patients in this group was 82 %, which is considered a statistically unfavorable group for breast cancer (stages T1–2, N0).Conclusion. Our findings suggest high accuracy of our method of breast cancer prognosis based on the quantitative assessment of CD4+ T-lymphocytes in tumor stromal cells. It can be used for breast cancer prognosis in early stages (T1–2, N0).

Expressions of β-Tryptase and Chymase in Lung Mast Cells due to Anaphylactic Shock through Histopathological Appearance at Different Post-Mortem Intervals

Highlights: The post-mortem interval is related to tryptase and chymase expressions in anaphylactic shock incidence Forensic experts can utilize tryptase and chymase as markers of anaphylactic (non-anaphylactoid) shock that occurs in the lungs. Abstract: Anaphylactic shock is a hypersensitivity response, a commonly type I hypersensitivity involving immunoglobulin E (IgE). It is caused by an antigen-antibody reaction that occurs immediately after a sensitive antigen enters the circulation. Anaphylactic shock is a clinical manifestation of anaphylaxis that is distributive shock, characterized by hypotension due to sudden blood vessel vasodilation and accompanied by a collapse in blood circulation that can result in death. β-tryptase and mast cell chymase expressions in the lungs of histopathological specimens that had experienced anaphylactic shock were examined at different post-mortem intervals in this study. A completely randomized design (CRD) method was employed by collecting lung samples every three hours within 24 hours of death, and then preparing histopathological and immunohistochemical preparations. The mast cell tryptase and chymase expressions were counted and summed up in each field of view, and the average was calculated to represent each field of view. The univariate analysis yielded p-values of 0.008 at the 15-hour post-mortem interval, and 0.002 at the 12-hour post-mortem interval. It was concluded that tryptase and chymase can be utilized as markers of anaphylactic (non-anaphylactoid) shock in the lungs.

Meme Karsinomlarında ER, PR, c-erbB2, Ki67, E-Cadherin Ekspresyonları, Nottingham Histolojik Grade ve Bazı Klinik Parametreler Arasındaki İlişkilerin Değerlendirilmesi

Objective: In this study, it was aimed to evaluate the relationships between Estrogen receptor (ER), Progesterone receptor (PR), c-erbB2 (HER2), Ki67, E-Cadherin expressions, Nottingham histological grade and some clinical parameters in breast carcinomas. Materials and Methods: A total of 74 patients diagnosed with breast carcinoma (CA) in our pathology department between 2018-2019 were included in this study. Immunohistochemical preparations stained with ER, PR, HER2, Ki67 and E-Cadherin were evaluated and analyzed retrospectively. For ER and PR, ≥1% expression was considered as positive staining, and <1% was considered as negative staining. HER2 expression was scored as 0, 1, 2 and 3. Ki67 proliferation index was considered as low (<10%), intermediate (10-20%) and high risk (>20%). The data were analyzed with chi-square test. Results: HER2 score showed a statistically significant change according to ER status (p=0.010). HER2 score also showed a statistically significant change according to PR status (p=0.004). There was a significant correlation between Ki67 and histological stage (p<0.001). Conclusions: Detection of high Ki67 index in breast carcinomas is poor prognostic. Detection of ER and PR expression and no expression of HER2 are good prognostic indicators. Preanalytical and analytical processes should be followed meticulously by pathologists.

SYMBIOSIS OF MEDICAL TECHNOLOGIES AND ARTIFICIAL INTELLIGENCE: NEW OPPORTUNITIES IN ONCOLOGY

SYMBIOSIS OF MEDICAL TECHNOLOGIES AND ARTIFICIAL INTELLIGENCE: NEW OPPORTUNITIES IN ONCOLOGY

Expression of Stanniocalcin-1 as a Predictor of Recurrence in Breast Cancer Patients

Introduction: About 40% of breast cancer patients will experience a recurrence and the life expectancy will decrease from 95% to 40%. Stanniocalcin-1 is a biomarker that is thought to have potential as a risk factor for recurrence.
 Objective: To determine the role of Stanniocalcin-1 (STC1) expression as a risk factor for recurrence in breast cancer patients.
 Methods: Cross sectional observational study on breast cancer patients, assessed the clinicopathological character and seen whether the patient's condition had a recurrence. Immunohistochemical staining with STC1 antibody was performed on the patient's paraffin block. Reading of immunohistochemical preparations using the Allred score method by looking at 5 fields of view. Data analysis regarding the relationship between STC1 and recurrence was carried out using the Chi-Square test or Fisher's Exact test, then Logistic regression analysis was performed to determine the significant relationship between risk factors for recurrence.
 Results: Expression of STC1 was significantly different in patients who had recurrence and who did not experience recurrence (p= 0.000). There was a significant relationship between STC1 expression with age, tumor size, lymph node status, type of cancer, estrogen receptor status and stage at diagnosis. (p<0.005).
 Conclusion: It can be concluded that STC1 has a strong relationship with recurrence, so it has the potential to be used as a predictor of recurrence in breast cancer.

Экспериментальное обоснование антисурдитантного применения производного триазино-индола при воздействии шума высокой интенсивности

In the study on the model of acoustic trauma in experimental animals (mice), the otoprotective effect of the triazine-indole derivative was shown due to the influence on the expression level of hypoxia-inducible factor (HIF) in the Corti organ and spiral ganglion cells. For therapeutic purposes, the drug was administered as a 1% solution at a dosage of 5, 7, 10 mg/kg 2 hours after acoustic exposure. For preventive purposes, the drug was administered at a dosage of 5, 7, 10 mg/kg for 3 days before acoustic exposure. The comparator drug was Cytoflavin. The control group received a saline solution. The immunohistochemical study was carried out using an anti-mouse polymerized immunohistochemical imaging system and mouse monoclonal antibodies to HIF1. In immunohistochemical preparations scanned using a digital scanner, the HIF1-positive area (%) in the cochlea and spiral ganglion cells was calculated using a morphometric program. The color was evaluated by the average cytochemical coefficient. It was found that the severity of HIF expression depended on the dose of the administered drug and coincided with electrophysiological changes in experimental animals. The antisurditant effect of the triazine-indole derivative is realized by influencing the level of HIF expression, which allows it to be regarded as a target molecule when using the drug.

The role of mismatch repair proteins expression in colorectal cancer

In the present study, carried out during the internship in Surgical Pathology of Santo Spirito Hospital in Casale Monferrato, a score based on the quantification of mismatch repair proteins (MMR) in patients with colorectal adenocarcinoma was devised, to determine whether a different expression in these proteins could correlate with some morphological parameters, and therefore whether it was possible to define a prognostic stratification for these patients. Objective During this period, 73 cases of colorectal adenocarcinoma referred from 01/01/2020 to 31/03/2021 were analyzed. The cases were investigated by immunohistochemical examination to assess the expression of mismatch repair proteins. Subsequently, the immunohistochemical preparations were observed by light microscopy, evaluating a total percentage of expression in the tumor tissue, in order to perform a correlation study between the expression of mismatch repair proteins and some morphological characteristics of the tumor at diagnosis. The total expression of MLH1 protein, as representative of the MLH1/PMS2 heterodimer, and of MSH6 protein, as representative of the MSH2/MSH6 heterodimer, was quantified. Methodology Evaluation of microsatellite instability was obtained by immunohistochemical investigation using ROCHE's automated VENTANA Bench-Mark Ultra platform. Correlation study, between tumor morphological data at diagnosis and expression of mismatch repair proteins, was performed by use of parametric and nonparametric tests.Results Total expression of MSH6 protein was higher than MLH1 expression in 60 of 73 cases. MSH6 protein is more frequently associated with high expression (from 75% to 100%) than MLH1, which is characterized by a lower expression range. The correlation test of MLH1 expression per- centage with perineural invasion gave a statistically significant results, with a p-value below the limit of significance (p=0.007191). In the case of MSH6, statistically significant p-values were shown for tumor budding (p=0.02577), perineural invasion (p=0.007498) and growth margin (p=0.04664). Conclusions Quantifying MMR protein expression in MSS tumors could be a valuable aid in therapeutic choice, as well as in prognostic stratification of MSS patients between subjects with high expression, and thus less aggressive tumors, and subjects with low expression lesions of MLH1 and/or MSH6, and thus more biologically aggressive tumors. If a different expression in mismatch repair proteins correlates with morphological characteristics of greater or lesser malignancy, it could be hypothesized that patients with MSS colorectal cancer, treated with the same therapy, may have a greater or lesser response, depending on the percentage of expression of mismatch repair proteins, estimating a more effective adjuvant therapeutic response in subjects with high expression.

Morphometric Parameters of Placental Villi in Parturient Women with COVID-19.

We performed a comparative morphological analysis of placental villi in parturient women with mild and moderate COVID-19 infection. The area and perimeter of terminal villi, their capillaries, and syncytiotrophoblast were assessed on immunohistochemical preparations with antibodies to CD31 using an image analysis system; the parameters of fetal vascular component in the placental villi were also assessed. Changes in the studied parameters differed in parturient women with mild and moderate COVID-19 infection. The observed increase in the total perimeter with a simultaneous decrease in the total capillary area and the degree of vascularization of the placental villi in parturient women with COVID-19 indicates impairment of circulation in the fetal compartment and the development of placental hypoxia, which can be the cause of unfavorable neonatal outcomes.

Структурна трансформація протокової системи підшлункової залози у хворих на хронічний панкреатит

Актуальність. Клітини проток становлять до 30 % від маси підшлункової залози (ПЗ) людини, а збільшення кількості бранчів проток відноситься до одних із перших сонологічних ознак хронічного панкреатиту (ХП). Мета дослідження — визначення морфологічних й імуногістохімічних особливостей протокової системи ПЗ хворих на ХП, яким були виконані резекційні та дренуючі операції з приводу ускладнень хронічного панкреатиту. Матеріали та методи. Проведено імуногістохімічне дослідження операційного матеріалу 40 хворих з ускладненим хронічним панкреатитом з використанням реакцій на a-ізоформу гладком’язового актину (a-SMA), десмін, фібронектин й колаген I типу. Результати. Характерна морфологічна картина розвитку ХП є поєднанням фіброзування з деформацією протокової системи за рахунок перидуктального фіброзу з розширенням міждолькових фіброзних септ, потовщенням інтралобулярних сполучно­тканинних тяжів, а також значною звивістістю і розширенням проток з ретенційними кістами. Значна структурна перебудова протокової системи ПЗ у хворих на ХП спостерігалася в 87,5 % випадків, в 17,5 % випадків картина доповнювалася явищами ацинарно-протокової метаплазії з трансформацією ацинарних клітин у протокові і формуванням дрібних дуктулоподібних структур, так званих тубулярних комплексів. У 7,5 % випадків в головній панкреатичній протоці визначалися вогнища атрофії протокового епітелію, які чергувалися з вогнищами папілярної інтраепітеліальної гіперплазії епітелію (PanIN). Розвитку панкреатичного фіброзу при ХП сприяє накопичення a-SMA+-активованих панкреатичних зірчастих клітин, що синтезують надлишок компонентів екстрацелюлярного матриксу, зокрема колагену I типу. Середня площа колагену І типу та інтенсивність його експресії були вірогідно нижче при зниженій експресії a-SMA (p < 0,05) і вірогідно вище у випадках вираженої експресії фібронектину (p < 0,05). Висновки. Вивчення структурних змін протокової системи ПЗ з використанням методів імуногістохімії є одним із способів покращення ранньої діагностики ускладнень ХП.

Muscle-nerve communication and the molecular assessment of human skeletal muscle denervation with aging.

Muscle fiber denervation is a major contributor to the decline in physical function observed with aging. Denervation can occur through breakdown of the neuromuscular junctions (NMJ) itself, affecting only that particular fiber, or through the death of a motor neuron, which can lead to a loss of all the muscle fibers in that motor unit. In this review, we discuss the muscle-nerve relationship, where signaling from both the motor neuron and the muscle fiber is required for maximal preservation of neuromuscular function in old age. Physical activity is likely to be the most important single factor that can contribute to this preservation. Furthermore, we propose that inactivity is not an innocent bystander, but plays an active role in denervation through the production of signals hostile to neuron survival. Investigating denervation in human muscle tissue samples is challenging due to the shared protein profile of regenerating and denervated muscle fibers. In this review, we provide a detailed overview of the key traits observed in immunohistochemical preparations of muscle biopsies from healthy, young, and elderly individuals. Overall, a combination of assessing tissue samples, circulating biomarkers, and electrophysiological assessments in humans will prove fruitful in the quest to gain more understanding of denervation of skeletal muscle. In addition, cell culture models represent a valuable tool in the search for key signaling factors exchanged between muscle and nerve, and which exercise has the capacity to alter.

Dysplastic Gangliocytoma: A Rare Example of Cerebellar Tumor with An Evident Genetic Profile (Cowden Syndrome)

Objective: To describe the histological and immunohistochemical findings observed when studying a dysplastic cerebellar gangliocytoma observed in a 33-year-old man with a history of Thyroid Cancer. Material and Methods: Radiological images (MRI) and histological and immunohistochemical preparations obtained from the cerebellar tissue were received in our laboratory. Results: A neoplasm constituted by aberrant-looking mature neurons was observed that showed negativity for the immunohistochemical markers of the PTEN protein, expression of the activity of the PTEN gene. Conclusions: Verify the diagnosis of a rare entity, clearly related in scientific publications with Cowden’s Syndrome.

E-cadherin expression in tumor emboli in gastric cancer

To determine the level of E-cadherin expression in tumor emboli, to compare it with expression in a tumor, to determine the dependence of E-cadherin expression in tumor emboli on the clinical and morphological characteristics of gastric cancer. We used samples of surgical material from 280 patients with a verified diagnosis of gastric cancer. E-cadherin expression was determined by immunohistochemical method. The results of the reactions were assessed semi-quantitatively and compared with the main clinical and morphological characteristics of gastric cancer (histological type according to the WHO classification 2019, histological type according to the classification of P. Lauren, clinical stage, depth of invasion (T), number of metastases in lymph nodes (N), presence or/absence of distant metastases (M), tumor localization in the stomach). Among 280 cases of cancer, emboli were detected only in 67 cases, used for further analysis. The rest of the samples were excluded from the analysis, since emboli did not get into the sections during the cutting of immunohistochemical preparations. The expression of E-cadherin in tumor emboli was significantly higher (p<0.001) than in tumor tissue. At the same time, no cases identified where the level of E-cadherin decreased in emboli compared to the tumor. A significant increase in the expression of E-cadherin in tumor emboli compared to the primary tumor was noted for all histological types according to WHO 2019, for intermediate and diffuse types according to the P. Lauren classification (p<0.001). Comparison of expression in emboli and tumors for neoplasms with different depths of invasion (T), different stages and different localizations did not reveal statistically significant differences. An increase in the expression of E-cadherin in emboli compared to tumors was characterized by a higher level of significance in the presence of metastases (N1, N2, N3a, N3b; p<0.001) than in the absence of metastases (N0; p=0.016). The study revealed a statistically significant increase in the expression of E-cadherin in tumor emboli compared to the primary tumor, which is evidence of its important role in maintaining the integrity of emboli and tumor dissemination.

Abstract PR08: Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas

Abstract Our study aims to establish whether CXorf67 has a role in the pathogenesis of posterior fossa (PF) ependymoma. Among molecular groups of ependymoma defined by DNA methylation profiling, PF type-A (PFA) is the commonest. PFA ependymomas present mainly in infants and are difficult to treat, having a 10-year overall survival of approximately 40%. Published genomic studies have not identified a recurrent driver mutation in PFA ependymomas, but they do show widespread epigenetic alterations, including global loss of histone H3 K27-trimethlyation (H3K27-me3). Another childhood PF tumor, the diffuse pontine glioma (DPG), also shows loss of H3K27-me3. In DPGs, loss of H3K27-me3 is associated with H3 K27M mutation, but the mechanism in PFA ependymomas is not yet established. We reexamined published PF ependymoma sequencing data and discovered recurrent mutations in a novel gene, CXorf67. Targeted sequencing in a series of PFA ependymomas revealed CXorf67 mutations in 22/234 (9.4%). CXorf67 is a single-exon gene of unknown function. Its protein product is predicted to be “disordered,” apart from one region towards the N terminus. Mutations in PFA ependymomas are missense, and there is a mutation hotspot in the “ordered” region. CXorf67 mutations are not present in other molecular groups of ependymoma and are rare in other cancers. Analyzing the DNA methylation profiles of 675 PFA ependymomas, we have discovered two subgroups, PFA-1 and PFA-2, and nine subtypes among these two subgroups. All PFA subtypes harbor CXorf67 mutations with the exception of PFA-1f and PFA-2c. Targeted sequencing in our tumor series also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. Two thirds of mutations in HIST1H3B, HIST1H3C, and H3F3A were found in PFA-1f ependymomas, among which H3 mutations were present at a frequency of 35%. Mutations in H3 genes and CXorf67 were mutually exclusive across our series of PFA ependymomas. We used Affymetrix u133v2 arrays to establish that CXorf67 is expressed at high levels in PFA ependymomas, in contrast to low levels in other ependymomas from the PF and supratentorial compartments. A mechanism for CXorf67 overexpression was revealed in a similar comparative analysis of CpG island methylation profiles, which showed that the promoter region of CXorf67 is hypomethylated in PFA tumors, but not in other ependymomas. Using immunohistochemical preparations, we detected expression of CXorf67 at the protein level in the nuclei of PFA ependymomas; PFB and supratentorial tumors were immunonegative. CXorf67 overexpression is found in all PFA molecular subtypes, except PFA-1f, which shows similar levels to those in supratentorial and PFB ependymomas. CXorf67 expression is unrelated to mutation status. Elevated CXorf67 expression is found in the Daoy and U2-OS cancer cell lines. We used immunoprecipitation (IP)/mass spectrometry (MS) to study proteins bound to CXorf67 in Daoy and U2-OS. Analysis of enriched peptides following immunoprecipitation of CXorf67 indicated that it binds EZH2, SUZ12, and EED, three core components of the PRC2 complex. Complementary immunoprecipitation of SUZ12 detected CXorf67. Detecting CXorf67 mutations in almost 10% of PFA ependymomas led to the discovery of overexpression related to promoter region hypomethylation in these tumors. Overexpression of CXorf67 was detected in all PFA ependymoma subtypes except PFA-1f, 35% of which harbor an H3 K27M mutation. The protein product of CXorf67 is found in tumor cell nuclei, where our IP/MS data suggest it is bound to PRC2. Our findings suggest that global loss of H3K27-me3 in PFA ependymomas could be related to overexpression of CXorf67 and its interaction with PRC2, except in tumors where alterations in histone H3 genes are responsible. However, the selective advantage of CXorf67 mutation is yet to be explained. Citation Format: Ji Wen, Jens-Martin Hübner, Wilda Orisme, Gang Wu, Bo Tang, Sujuan Jia, John Easton, Kelly Haupfear, Brian D. Freibaum, Hong Joo Kim, Anthony High, Junmin Peng, Ruth G. Tatevossian, J. Paul Taylor, Stefan M. Pfister, Jinghui Zhang, Kristian W. Pajtler, Marcel Kool, David W. Ellison. Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR08.

Diffusion-Weighted MRI Is Insensitive to Changes in the Tumor Microenvironment Induced by Antiangiogenic Therapy

Antiangiogenic treatment (AAT) used in combination with radiation therapy or chemotherapy is a promising strategy for the treatment of several cancer diseases. The vascularity and oxygenation of tumors may be changed significantly by AAT, and consequently, a noninvasive method for monitoring AAT-induced changes in these microenvironmental parameters is needed. The purpose of this study was to evaluate the potential usefulness of diffusion-weighted magnetic resonance imaging (DW-MRI). DW-MRI was conducted with a Bruker Biospec 7.05-T scanner using four diffusion weightings and diffusion sensitization gradients in three orthogonal directions. Maps of the apparent diffusion coefficient (ADC) were calculated by using a monoexponential diffusion model. Two cervical carcinoma xenograft models (BK-12, HL-16) were treated with bevacizumab, and two pancreatic carcinoma xenograft models (BxPC-3, Panc-1) were treated with sunitinib. Pimonidazole and CD31 were used as markers of hypoxia and blood vessels, respectively, and fraction of hypoxic tissue (HFPim) and microvascular density (MVD) were quantified by analyzing immunohistochemical preparations. MVD decreased significantly after AAT in BK-12, HL-16, and BxPC-3 tumors, and this decrease was sufficiently large to cause a significant increase in HFPim in BK-12 and BxPC-3 tumors. The ADC maps of treated tumors and untreated control tumors were not significantly different in any of these three tumor models, suggesting that the AAT-induced microenvironmental changes were not detectable by DW-MRI. DW-MRI is insensitive to changes in tumor vascularity and oxygenation induced by bevacizumab or sunitinib treatment.

DCE-MRI of Sunitinib-Induced Changes in Tumor Microvasculature and Hypoxia: A Study of Pancreatic Ductal Adenocarcinoma Xenografts

The purpose of this study was dual: to investigate (a) whether sunitinib may induce changes in tumor microvasculature and hypoxia in pancreatic ductal adenocarcinoma (PDAC) and (b) whether any changes can be detected by DCE-MRI. Sunitinib-treated and untreated control tumors of two PDAC xenograft models (BxPC-3 and Panc-1) were subjected to DCE-MRI before the imaged tumors were prepared for quantitative analysis of immunohistochemical preparations. Pimonidazole was used as a hypoxia marker, and fraction of hypoxic tissue (HFPim), density of CD31-positive microvessels (MVDCD31), and density of αSMA-positive microvessels (MVDαSMA) were measured. Parametric images of Ktrans and ve were derived from the DCE-MRI data by using the Tofts pharmacokinetic model. BxPC-3 tumors showed increased HFPim, decreased MVDCD31, unchanged MVDαSMA, and increased vessel maturation index (VMI = MVDαSMA/MVDCD31) after sunitinib treatment. The increase in VMI was seen because sunitinib induced selective pruning rather than maturation of αSMA-negative microvessels. Even though the microvessels in sunitinib-treated tumors were less abnormal than those in untreated tumors, this microvessel normalization did not improve the function of the microvascular network or normalize the tumor microenvironment. In Panc-1 tumors, HFPim, MVDCD31, MVDαSMA, and VMI were unchanged after sunitinib treatment. Median Ktrans increased with increasing MVDCD31 and decreased with increasing HFPim, and the correlations were similar for treated and untreated BXPC-3 and Panc-1 tumors. These observations suggest that sunitinib may induce significant changes in the microenvironment of PDACs, and furthermore, that Ktrans may be an adequate measure of tumor vascular density and hypoxia in untreated as well as sunitinib-treated PDACs.

COMPARISON OF THE PROTECTIVE ROLE OF RANITIDINE AND TWO MEDICINAL PLANTS (GINGER AND PEPPERMINT) AGAINST INDOMETHACIN-INDUCED GASTRIC ULCER IN ALBINO RATS: A HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY

The aim of the present study is to compare the gastroprotective potential of two medicinal plants, namely ginger and peppermint, versus the anti-ulcer drug (ranitidine), against indomethacin-induced gastric ulcer in rats. Thirty adult male rats were divided equally into five groups. Group I: (normal control) animals received distilled water. Group II animals received indomethacin to induce ulcer. Group III: animals were given ranitidine followed by indomethacin. Group IV: animals were given ginger juice followed by indomethacin. Group V: animals were also treated with peppermint juice prior to indomethacin. Animals of all groups received the treatments orally. Rats were sacrificed 7hr later, and their stomachs were isolated for macroscopical and microscopical examinations. The results revealed a significant (p < 0.01) reduction of the ulcerated surface in ranitidine, ginger and peppermint pretreated rats, when compared to the ulcer control group. Indomethacin-treated rats showed focal disruption of the gastric mucosa and the cells lining the damaged gastric glands appeared with deeply acidophilic cytoplasm and pyknotic nuclei. Submucosal oedema, inflammatory cell infiltration, and dilated congested blood vessels were also evident. In periodic acid Schiff’s (PAS) reaction, surface columnar epithelial (mucous) cells and mucous neck cells exhibited a weak PAS-positive reaction compared to normal control group. In immunohistochemical preparations, proliferating cell nuclear antigen (PCNA) expression was reduced in the ulcerated areas by indomethacin. Alternatively, ginger or peppermint-pretreated animals, similar to the ranitidine-pretreated animals, showed less gastric damage compared to the ulcer control group. This study demonstrates that comparable to ranitidine, ginger and peppermint showed gastroprotective efficacy against indomethacin-induced peptic ulcer in rats.

Introductory Teaching Tool Utilizing Immunohistochemistry to Explain the Placenta

Introduction: The placenta occupies an important place in science, medicine, and law; yet, it remains a difficult organ to explain and understand because of its unique characteristics. Methods and Materials: A descriptive observational study was carried out on placentas. Placental components were illustrated with diagrams and corresponding microphotographs were highlighted by immunohistochemical staining to identify cell-types and structures. Results: The placenta was diagrammatically demonstrated by dividing it into chorionic plate with umbilical cord, basal plate and placental disc. In highlighting the histological components, approximately 80 immunohistochemical preparations were taken into account Conclusion: Placental complexity continues to be the critical interface for maternal-fetal dialogue. Better educational methods are needed to explain the complex pathophysiology of this essential organ. Improving placental education is the key to attracting young researchers dedicated to this field.

Abstract 1801: Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts

Abstract Background: Studies comparing the effect of antiangiogenic agents targeting different angiogenic pathways are sparse. The purpose of this study was to compare the effect of properdistatin and sunitinib treatment in a preclinical model of malignant melanoma. Properdistatin is a small peptide derived from the thrombospondin-1 domain of the plasma protein properdin, and sunitinib is a tyrosine kinase inhibitor targeting several receptors including the vascular endothelial growth factor receptors. Methods: R-18 human melanoma xenografts growing in dorsal window chambers were treated with properdistatin, sunitinib, or vehicle. Parameters describing the morphology of tumor vasculature were assessed from high-resolution transillumination images, and BST (blood supply time; the time needed for arterial blood to flow from the main supplying artery to downstream microvessels) was assessed from first-pass imaging movies recorded after a bolus of fluorescence-labeled dextran had been administered intravenously. Tumor hypoxia was assessed from immunohistochemical preparations of the imaged tissue by using pimonidazole as a hypoxia marker. Results: Properdistatin treatment selectively removed small-diameter vessels and reduced BST, whereas sunitinib treatment reduced the density of small- and large-diameter vessel similarly and did not change BST. These observations imply that properdistatin treatment reduced geometric resistance to blood flow and improved vascular function, whereas sunitinib treatment did not affect vascular function. Accordingly, sunitinib-treated tumors showed higher hypoxic fractions than properdistatin-treated tumors. Conclusions: Properdistatin and sunitinib both inhibited angiogenesis, but had distinctly different effects on vascular morphology, vascular function, and extent of hypoxia in R-18 human melanoma xenografts. Citation Format: Jon-Vidar Gaustad, Trude G. Simonsen, Lise Mari K. Andersen, Einar K. Rofstad. Antiangiogenic agents targeting different angiogenic pathways have opposite effects on tumor hypoxia in R-18 human melanoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1801. doi:10.1158/1538-7445.AM2017-1801