Conventional Hematoxylin-eosin Staining Research Articles

Abstract 3768: Optimization of digital pathology through laser capture microdissection with a 405 nm laser

Abstract Tumor cellular heterogeneity is a complex problem in cancer molecular diagnostics and personalized therapy. The tumor is a product of the different types and interactions of host and immune cells. Investigators have created a variety of methods to procure separate subpopulations of the tumor microenvironment for individual analysis. One of the most successful methods of this is laser capture microdissection (LCM). This method captures specific subpopulations of cells under direct microscopic visualization. This LCM technology has successfully been used for over 20 years, revealing a variety of insights into cancer pathogenesis and mechanisms of therapeutic response employed in numerous clinical trials. Nevertheless, there are two drawbacks to the current systems. For UV cutting (365 nm), the tissue is significantly damaged by the UV light energy. Infrared (808 nm) laser systems do not damage the tissue however, this method has low power and poor resolution. Our first hypothesis to be tested is that a new class of near UV 405 nm laser will have small capture areas approaching single cells while simultaneously preventing cellular damage. The second weakness of LCM is the requirement for an open-faced tissue section without a coverslip. Moreover, when visualizing the tissue of interest, the identification of regions of interest is obscured due to the refractive index mismatch between the laser microdissection cap and the tissue surface. We hypothesize that using a volatile organic compound with a refractive index similar to glass would elucidate the tissue region of interest to allow proper visualization. We successfully developed and tested a near 405 nm wavelength LCM system that utilizes a “liquid” coverslip to visualize the sample of interest with higher clarity, resolution, and contrast for immunohistochemistry, immuno-fluorescence, blood smear slides, and conventional hematoxylin-eosin tissue staining. Overall, these critical improvements LCM technology now allow for single-cell tissue capture, system automation, tissue visualization, and opens the door for artificial intelligence-assisted spatial profiling. Citation Format: Thomas Philipson, Marissa Howard, Kevin Johnson, Amanda Still, Furkat Yunusov, Emanuel Petricoin, Virginia Espina, Noel Gonzalez, Alan Carpino, Lance Liotta. Optimization of digital pathology through laser capture microdissection with a 405 nm laser [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3768.

A diagnostic strategy for pulmonary fat embolism based on routine H&E staining using computational pathology.

Pulmonary fat embolism (PFE) as a cause of death often occurs in trauma cases such as fractures and soft tissue contusions. Traditional PFE diagnosis relies on subjective methods and special stains like oil red O. This study utilizes computational pathology, combining digital pathology and deep learning algorithms, to precisely quantify fat emboli in whole slide images using conventional hematoxylin-eosin (H&E) staining. The results demonstrate deep learning's ability to identify fat droplet morphology in lung microvessels, achieving an area under the receiver operating characteristic (ROC) curve (AUC) of 0.98. The AI-quantified fat globules generally matched the Falzi scoring system with oil red O staining. The relative quantity of fat emboli against lung area was calculated by the algorithm, determining a diagnostic threshold of 8.275% for fatal PFE. A diagnostic strategy based on this threshold achieved a high AUC of 0.984, similar to manual identification with special stains but surpassing H&E staining. This demonstrates computational pathology's potential as an affordable, rapid, and precise method for fatal PFE diagnosis in forensic practice.

Conventional HE staining combined with two special staining methods to identify antemortem stab wound in case with highly decomposed mutilated corpses.

This study aims to identify an antemortem neck stab wound on a highly decomposed, headless and mutilated body by conventional hematoxylin-eosin (HE) staining combined with Ponceau-Victoria blue B staining (P-VB staining) and Masson staining. Specifically, a tissue sample was excised from the skin and muscle tissue at the junction of the normal and brownish discolored areas around the suspected stabbing tract of the left neck, in the upper and lower wound-clavicle-shoulder region. Conventional HE staining only provides a morphological and structural outline of the tissue, with both the injury hemorrhage and local connective tissue appearing eosinophilic pink. However, P-VB staining shows obvious contrast between the injury hemorrhage and connective tissue, with the former appearing yellow-green and the latter appearing orange-red. Similarly, Masson staining of the injury hemorrhage and connective tissue contrast clearly with purple-red and dark blue, respectively. Therefore, our study highlights that conventional HE staining with the combination of P-VB staining and Masson staining allowed for a clearer and corroborated identification of antemortem injury and hemorrhage from the stab wound in highly decomposed mutilated corpses.

Unpaired virtual histological staining using prior-guided generative adversarial networks.

Fibrosis is an inevitable stage in the development of chronic liver disease and has an irreplaceable role in characterizing the degree of progression of chronic liver disease. Histopathological diagnosis is the gold standard for the interpretation of fibrosis parameters. Conventional hematoxylin-eosin (H&E) staining can only reflect the gross structure of the tissue and the distribution of hepatocytes, while Masson trichrome can highlight specific types of collagen fiber structure, thus providing the necessary structural information for fibrosis scoring. However, the expensive costs of time, economy, and patient specimens as well as the non-uniform preparation and staining process make the conversion of existing H&E staining into virtual Masson trichrome staining a solution for fibrosis evaluation. Existing translation approaches fail to extract fiber features accurately enough, and the decoder of staining is unable to converge due to the inconsistent color of physical staining. In this work, we propose a prior-guided generative adversarial network, based on unpaired data for effective Masson trichrome stained image generation from the corresponding H&E stained image. Conducted on a small training set, our method takes full advantage of prior knowledge to set up better constraints on both the encoder and the decoder. Experiments indicate the superior performance of our method that surpasses the previous approaches. For various liver diseases, our results demonstrate a high correlation between the staging of real and virtual stains (ρ=0.82; 95% CI: 0.73-0.89). In addition, our finetuning strategy is able to standardize the staining color and release the memory and computational burden, which can be employed in clinical assessment.

Immunohistochemical Analysis of CYP11B2, CYP11B1 and β-catenin Helps Subtyping and Relates With Clinical Characteristics of Unilateral Primary Aldosteronism.

Background: Primary aldosteronism is caused by aldosterone overproduction. While conventional hematoxylin-eosin staining can demonstrate morphological abnormality, it cannot provide any functional histopathological information. We aimed to identify the diagnostic, functional and prognostic value of CYP11B2, CYP11B1, and β-catenin immunostaining in unilateral hyperaldosteronism. Method: A total of 134 patients with unilateral hyperaldosteronism were recruited in our study. The expression of CYP11B2, CYP11B1, and β-catenin was evaluated semiquantitatively on 134 patients’ sections using immunohistochemistry technology and the relationship with clinical data was assessed. Results: Patients were classified into four subtypes based on CYP11B2 staining as below: (1)118 patients with unilateral single aldosterone-producing adenoma (APA), (2)11 with unilateral multiple APA, (3)four with aldosterone-producing cell cluster (APCC), and (4)one with an undefined source. Adjusted CYP11B2 H-score was correlated with serum aldosterone, aldosterone to renin ratio (ARR), and serum potassium. In the abnormal β-catenin staining group, hypertension duration, aldosterone, ARR, cortisol, tumor diameter, tumor area, and CYP11B2 H-score were significantly higher than those of the wild-type group. Serum potassium level was significantly lower in the abnormal β-catenin staining group. Age, gender, BMI, family history of hypertension, adjusted CYP11B2 and CYP11B1 H-scores differed significantly between complete clinical success and incomplete clinical success groups. Age, gender and family history of hypertension were independently associated with complete clinical success based on multivariate logistic regression analysis. Conclusion: CYP11B2 immunostaining could improve the differential diagnosis of unilateral hyperaldosteronism. Adjusted CYP11B2 H-score could be used as a histopathological marker to reflect the severity of unilateral APA. Dysregulation of Wnt/β-catenin signaling and impaired β-catenin degradation may provoke the proliferation and enhance the steroidogenic ability of APA tumor cells, indicating that the Wnt pathway might be a potential, actionable, therapeutic target in the treatment of hyperaldosteronism. Age, sex and family history of hypertension were independent predictors of clinical outcome after adrenalectomy for unilateral hyperaldosteronism.

Histological Evidence of Multiple Spawning in Wild Female Japanese Eel Anguilla japonica.

The post ovulatory follicle (POF) is an important and reliable tissue structure used to investigate the spawning history in teleost fish. Fresh POFs shortly after spawning are comprised of cellular (follicular cells) and acellular (basement membrane and fibrils such as elastic fibers) components. The cellular components are quickly disintegrated by means of apoptosis, while the acellular components persist for a longer period. Since cellular components are well visualized by conventional hematoxylin-eosin (HE) staining but acellular components are not stained well, old POFs that have lost cellular components are difficult to identify. In this study, periodic acid-Schiff and Victoria blue staining, which can distinctly visualize acellular POF components, were applied to the ovarian tissues of Japanese eel (Anguilla japonica) (n = 9) captured from June to August of 2008, 2009, and 2013 at the southern West Mariana Ridge, a spawning area for Japanese eels. Only new POFs were observed in seven females caught in June, and these females had ovaries with early-to mid-vitellogenic stage oocytes. Both fresh and old POFs were observed in a female caught in July, and only mid-vitellogenic stage oocytes were observed. Only old POFs and no vitellogenic stage oocyte were observed in a female caught in August. A progressive decrease in muscle lipid content, gonad somatic index, and condition factors was observed from June to August. Thus, the female Japanese eel can spawn at least twice or three times at most during spawning season, depending on energy reserve.

The Prognostic Significance of Peritumoral Lymphocytes' Band-like Structure in Type II Endometrial Cancer.

Peritumoral ectopic lymphoid-like structures and tertiary lymphoid structures (TLS), have been identified in various cancers. However, evidence for the role of TLS in endometrial cancer (EC) is lacking. We found a cluster of peritumoral lymphocytes with band-like structures (PLB) in the forefront of EC and analyzed their association with the clinical outcome. This was a single-center, retrospective cohort study. We evaluated peritumoral lymphoid cells using conventional hematoxylin-eosin and immunohistochemical staining by semi-quantitative digital analysis. A total of 85 cases were included and the presence of PLB was examined. A strong correlation was observed between the density of PLB and progression-free survival (very low, low vs. intermediate, high; HR=0.22; 95%CI=0.093-0.52; p<0.001) and overall survival (very low, low vs. intermediate, high; HR=0.259; 95%CI=0.091-0.73; p=0.011). PLB in type II EC show a strong association with fatal outcome.

Effect of benazepril on nuclear factor E2 related factor 2, nicotinamide adenine dinucleotide phosphate oxidase and reactive oxygen species in rats with hepatic fibrosis

Objective: To study the effect of benazepril on the expression of nuclear factor E2 related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and reactive oxygen species (ROS) concentration in rats with hepatic fibrosis and to explore the possible antifibrotic mechanism of benazepril. Methods: Twenty-two healthy male Sprague-Dawley rats were randomly divided into 3 groups: control group (6 rats), model group (8 rats) and benazepril treatment group (8 rats). Two rats died during modeling and treatment in the model group and the benazepril treatment group, and a model of hepatic fibrosis induced by carbon tetrachloride (CCL(4)) was established. The rats in benazepril group were given benazepril for 8 weeks by gastric gavage. The assessment of liver tissue damage in each group was measured using conventional hematoxylin-eosin and Masson staining. The mRNA level of Nrf2, NOX4 in liver tissue was detected by RT-PCR, and serum ROS concentration was determined by colorimetry. All data were expressed in mean ± standard deviations, and were analyzed using SPSS21.0 statistical software. The data were compared using one-way analysis of variance, and the LSD-t method was used for pairwise comparison between the two groups. The correlation analysis was performed by Spearman's correlation analysis. Results: In the liver of the model group, with the aggravation of liver fibrosis the expression of Nrf2mRNA, NOX4 mRNA and ROS concentration were higher than control group [(4.01 ± 3.40), (31.78 ± 3.96), (1.82 ± 0.46) μg/ ml vs. (0.12 ± 0.11), (2.03 ± 0.31), (1.56±0.84) μg/ml, P < 0.05]. After benazepril treatment, NOX4 mRNA expression and ROS concentration were decreased than the model group [(15.93 ± 5.01), (0.78 ± 0.44) μg/ml vs. (31.78 ± 3.96), (1.82 ± 0.46) μg /ml, P < 0.05], while Nrf2 mRNA expression was higher than the model group [(6.69 ± 4.86) vs. (4.01 ± 3.40), P < 0.05]. There was a positive correlation between Nrf2 and NOX4, Nrf2 and ROS, and NOX4 and ROS (r = 0.616, 0.411, 0.802, P < 0.05). Conclusion: Benazepril may exert an anti-hepatic fibrosis effect by activating Nrf2 expression, or may inhibit the ROS-mediated oxidative stress in response to NOX4.

Comparison of artificial dermis Lando® versus Pelnac® combined with induced membrane technique in treatment of composite trauma in rabbit bilateral femurs

Objective To compare 2 kinds of artificial dermis Lando® versus Pelnac® combined with induced membrane technique in the treatment of composite trauma in rabbit bilateral femurs. Methods Twenty-seven male rabbits, weighing from 1.92 kg to 2.21 kg (average, 2.04 kg), were used in the experiments. After models of composite trauma were created in the bilateral femurs of all the rabbits, the bilateral femurs of 9 random rabbits were subjected to the treatment of artificial dermis Lando® plus induced membrane (Lando® group), the bilateral femurs of another 9 random rabbits to the treatment of artificial dermis Pelnac® plus induced membrane (Pelnac® group), the left femur of the remaining 9 rabbits to the treatment of induced membrane (control group) and the right femur of the remaining 9 rabbits to no treatment (sham operation group). Three rabbits from each group (the same for the control and sham operation groups) were randomly sacrificed at 2, 4 and 6 weeks after operation. Samples from the operation sites were taken for gross observation of the induced membrane and observation of the microstructure of the membrane by conventional hematoxylin-eosin staining. In addition, the microvessel density (MVD) was counted under microscopy taking CD34 immunohistochemistry as the standard. The data were statistically analyzed. Results The collagen sponge layer was completely degraded 2 weeks after operation in the Lando® group but not in the Pelnac® group. The MVD [(0.90±0.55)/HPF] in the Lando® group was significantly greater than that in the Pelnac® group [(0.28±0.13)/HPF] (P 0.05). At 6 weeks postoperatively, the MVD [(4.97±0.76)/HPF] in the Lando® group was significantly smaller than that in the Pelnac® group [(7.06±1.03)/HPF] (P 0.05). The MVD at 4 and 6 weeks after operation in the control and the sham operation groups were all significantly different from those in the Lando® and the Pelnac® groups (P<0.05). Conclusions In the treatment of composite trauma in rabbits, the 2 kinds of artificial dermis combined with the induction membrane technique can lead to formation of induced membrane structure, providing a new alternative treatment for patients with bone and soft tissue defects caused by various causes in the clinic. The Lando® artificial dermis may accelerate the vascularization of induced membrane at 2 weeks while the Pelnac® may accelerate the vascularization at 4 and 6 weeks. Key words: Femur; Dermis; Soft tissue injuries; Artificial dermis; Bone defect

Effects of nebulized N--acetylcystein on the expression of HMGB1 and RAGE in rats with hyperoxia--induced lung injury.

To investigate the role of high mobility group box 1 (HMGB1) and receptor for advanced glycation end product (RAGE) in the lungs of hyperoxia-induced rats and the effect of N--acetlycystein (NAC). A model of hyperoxic lung injury was established, rats in the NAC intervention, and control, hyperoxia group were given nebulized NAC aerosol, nebulized same volume of saline once a day for 7 consecutive days, respectively. Wet/dry ( W/ D) ratio of the lungs was determined to evaluate the edema of the lung tissues. Conventional hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissues. Immunohistochemical staining was used to investigate the expression of HMGB1 and RAGE in the lung tissues. Quantitative reverse-transcription polymerase chain reaction and western blot analysis were used to measured the changes in the messenger RNA (mRNA) and protein expression of HMGB1 and RAGE, respectively. Weight gain of the rats in the hyperoxia group was significantly slower than that in the control group and intervention group (p < 0.05). HE staining results showed lung tissues in the hyperoxia group were severely damaged compared with control group. W/D ratio in hyperoxia group was significantly higher than that in control group and intervention group (p < 0.05). Protein and mRNA expression of HMGB1 and RAGE in the hyperoxia group were significantly higher than control group and intervention group (p < 0.05). HMGB1 and RAGE were involved in the pathogenesis of hyperoxia-induced lung injury, inhalation of NAC might alleviate hyperoxia-induced lung injury by regulating the expression of HMGB1 and RAGE.

Inhibitors of HIF-1α and CXCR4 Mitigate the Development of Radiation Necrosis in Mouse Brain

There is mounting evidence that, in addition to angiogenesis, hypoxia-induced inflammation via the hypoxia-inducible factor 1α (HIF-1α)-CXC chemokine receptor 4 (CXCR4) pathway may contribute to the pathogenesis of late-onset, irradiation-induced necrosis. This study investigates the mitigative efficacy of an HIF-1α inhibitor, topotecan, and a CXCR4 antagonist, AMD3100, on the development of radiation necrosis (RN) in an intracranial mouse model. Mice received a single-fraction, 50-Gy dose of hemispheric irradiation from the Leksell Gamma Knife Perfexion and were then treated with either topotecan, an HIF-1α inhibitor, from 1 to 12weeks after irradiation, or AMD3100, a CXCR4 antagonist, from 4 to 12weeks after irradiation. The onset and progression of RN were monitored longitudinally via noninvasive, invivo magnetic resonance imaging (MRI) from 4 to 12weeks after irradiation. Conventional hematoxylin-eosin staining and immunohistochemistry staining were performed to evaluate the treatment response. The progression of brain RN was significantly mitigated for mice treated with either topotecan or AMD3100 compared with control animals. MRI-derived lesion volumes were significantly smaller for both of the treated groups, and histologic findings correlated well with the MRI data. By hematoxylin-eosin staining, both treated groups demonstrated reduced irradiation-induced tissue damage compared with controls. Furthermore, immunohistochemistry results revealed that expression levels of vascular endothelial growth factor, CXC chemokine ligand 12, CD68, CD3, and tumor necrosis factor α in the lesion area were significantly lower in treated (topotecan or AMD3100) brains versus control brains, while ionized calcium-binding adapter molecule 1 (Iba1) and HIF-1α expression was similar, though somewhat reduced.CXCR4 expression was reduced only in topotecan-treated mice, while interleukin 6 expression was unaffected by either topotecan or AMD3100. By reducing inflammation, both topotecan and AMD3100 can, independently, mitigate the development of RN in the mouse brain. When combined with first-line, antiangiogenic treatment, anti-inflammation therapy may provide an adjuvant therapeutic strategy for clinical, postirradiation management of tumors, with additional benefits in the mitigation of RN development.

Clinicopathologic characteristics of HIV/AIDS-related plasmablastic lymphoma.

Plasmablastic lymphoma is a rare and aggressive B cell lymphoma that is considered to be strongly associated with HIV infection. This article explores the histological morphology and immunohistochemical characteristics of HIV/AIDS-related plasmablastic lymphoma with the goal of improving the diagnosis and treatment of this rare tumor. According to criteria of the World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues (2008), six plasmablastic lymphoma cases admitted to the Shanghai Public Health Clinical Center were comprehensively analyzed with conventional hematoxylin-eosin staining, immunohistochemical staining and in situ hybridization. The morphological features of six tumors were consistent with PBL. Immunohistochemical staining showed that all six cases were negative for CD19, CD20, and CD79a, and positive for OCT-2, BOB-1, VS38c, and melanoma ubiquitous mutated 1. The Ki67 proliferation index was higher than 90% in all six cases. In situ hybridization indicated that four cases were EBER-positive. In addition, three cases had C-MYC translocation rearrangement. Our results showed that the immunophenotypes of PBL vary, which makes PBL diagnosis difficult. Therefore, morphological characteristics, immunophenotypic markers, and clinical data should be used in combination to enable an accurate diagnosis, especially in the presence of immunophenotypic variation, as this approach will facilitate timely treatment.

A reliable scoring system after major liver resection in mice

BackgroundPosthepatectomy liver failure and its transplant counterpart, small-for-size syndrome, remain significant limitations for liver resections and segmental liver transplantation. Partial hepatectomy in mice is one of the most commonly used models to study liver regeneration, but blood and tissue sampling necessary to collect data can affect outcomes or even require euthanasia. We therefore developed a quantitative observational system to predict death from hepatectomy during the first 24 postoperative hours. Materials and methodsA total of 100 female, 10 to 12-week-old C57BL/6 mice underwent two-thirds hepatectomy and were monitored for up to 7 d. Our scoring system was based on five categories, each assigned 0-2 points: activity level, body posture, fur condition, respiratory status, and eye appearance. Seventy-five mice were scored 6 h, 12 h, 24 h, 2 d, 3 d, 5 d, and 7 d after surgery. The remaining 25 mice were scored similarly, but underwent, in addition, blood sampling for serum alanine aminotransferase, total bilirubin, interleukin-6, tumor necrosis factor-alpha, or euthanasia with liver sampling for conventional hematoxylin-eosin and Ki-67 staining. ResultsRetrospective analysis indicated that body condition scores ≤5 on two consecutive time points within the first 24 postoperative hours accurately predicted eventual death. Animals in the low scoring group also had significantly higher serum alanine aminotransferase, total bilirubin, interleukin-6, tumor necrosis factor-alpha, more hepatocyte necrosis in hematoxylin-eosin, and fewer Ki-67 positive hepatocytes. ConclusionsOur scoring system accurately predicts survival, hepatocyte damage, liver regeneration, and systemic inflammation in a mouse hepatectomy model, within the first 24 hours of surgery. This could be useful in evaluating posthepatectomy interventions for their effect on survival and liver regeneration.

Fetal Development of the Bovine Uterus: A Light Microscopy and Immunohistochemical Study

Important steps during the prenatal development of the bovine uterus are described using conventional hematoxylin-eosin staining of fetuses from different developmental stages [crown-rump length (CRL) 9.2-94.0 cm]. Additionally, a number of intermediate filaments (keratin 7, 8, 14, 18, 19; and vimentin), the basement membrane protein laminin, smooth-muscle marker (SMA), and S100 were studied to further characterize certain differentiation processes. During early development, the uterine epithelium is simple or (pseudo)stratified with bud-like protrusions. Developing caruncles can be observed in the corpus uteri at a CRL of 15.8 cm onwards, showing a simple, keratin-positive epithelium. In contrast, the intercaruncular areas are characterized by a (pseudo)stratified epithelium, which also shows positive staining in a different manner for the investigated keratins. A differentiation of smooth muscle cell layers can be observed from a CRL of 24.4 cm onwards. Intense SMA-positive cells/fibers, arranged perpendicularly to the developing circular SMA-positive muscle cell layer, can be found preferentially located in the developing caruncles. Lymphocytes occur in the uterine epithelium and stroma in the corpora and cornua of fetuses with a CLR of 15.8 cm and higher.

Oral paracoccidiodomycosis mimicking lip carcinoma

A 65-year-old man, actively engaged in farming activities, with a 90 pack-year smoking history, presented with a painless ulcerated lesion on the left labial commissure. He was referred to our department because of suspected neoplasia. The lesion progressed slowly through a 2-year period. Oral examination revealed an extensive ulcer, although flat and with no signs of orbicularis oris muscle infiltration (Fig. 1), involvement of mandibular division of facial nerve, or suspicious neck lymph nodes metastasis. A representative incisional biopsy was performed. Histopathological examination exhibited chronic granulomatous inflammation on conventional hematoxylin–eosin staining. Grocott-Gomori staining showed fungal structures. Clinical and pathological evidences supported the diagnosis of paracoccidioidomycosis (PCM). Itraconazole was maintained for six months with satisfactory response to proposed therapy. PCM is a systemic mycosis characterized by acute or chronic tissue inflammation caused by Paracoccidioides brasiliensis, a pathogenic thermally dimorphic fungus that is endemic to Latin America.1 Active agricultural laborers, masons and civil construction workers, or those who have

Detection of lymphovascular invasion in urothelial carcinoma of the bladder through D2-40 immunostaining

Lymphovascular invasion (LVI) represents a surrogate marker for micrometastatic urothelial carcinoma of the bladder (UCB). We evaluated whether D2-40 immunhistochemistry (IHC) alters detection of LVI when compared to conventional HE (hematoxylin-eosin) staining of UCB specimens in a blinded fashion. HE- and D2-40-IHC-stained representative sections of 80 patients after radical cystectomy (RC) were re-reviewed. LVI detection rates were recorded and compared after blinded evaluation. LVI was present in 53 patients (66.3%) in HE-stained sections and in 44 patients (55%) in D2-40 stainings. In 13 patients, LVI (16.3%) was found in HE stained sections but not confirmed when IHC was applied (false positive when using IHC as a reference standard). D2-40 IHC identified LVI in 4 additional patients (5%) who were classified as LVI negative in conventional HE staining (false negative). 52 patients (65%) were lymph node negative (pN0), 21 of whom (40.4%) were LVI positive in conventional HE sections and 16 of whom (30.8%) were LVI positive in IHC. In 9 pN0 patients (17.3%), LVI was diagnosed in HE sections but not confirmed by IHC (false positive). D2-40 IHC identified LVI in 4 additional patients (7.7%) who were node negative and classified as LVI negative in conventional HE staining (false negative). In patients who experienced recurrence (n=35) and who were classified as pN0 at the time of RC, HE staining resulted both in false-positive (n=2; 5.7%) and false-negative (n=3; 8.6%) findings. Different detection rates of LVI were observed when using IHC with D2-40 in UCB patients compared to conventional HE staining. The routine use of D2-40 IHC should be considered in clinical trial design to improve risk stratification of pN0 patients after RC.

Detection of Lymphovascular Invasion by D2-40 (Podoplanin) Immunoexpression in Endometrial Cancer

Lymph node involvement is a major feature in tumor spread of endometrial cancer and predicts prognosis. Therefore, evaluation of lymph vessel invasion (LVI) in tumor tissue as a predictor for lymph node metastasis is of great importance. Immunostaining of D2-40 (podoplanin), a specific marker for lymphatic endothelial cells, might be able to increase the detection rate of LVI compared with conventional hematoxylin-eosin (H-E) staining. The aim of this retrospective study was to analyze the eligibility of D2-40-based LVI evaluation for the prediction of lymph node metastases and patients' outcome. Immunohistochemical staining with D2-40 monoclonal antibodies was performed on paraffin-embedded tissue sections of 182 patients with primary endometrioid adenocarcinoma treated in 1 gynecologic cancer center. Tumors were screened for the presence of LVI. Correlations with clinicopathological features and clinical outcome were assessed. Immunostaining of D2-40 significantly increased the frequency LVI detection compared with conventional H-E staining. Lymph vessel invasion was identified by D2-40 in 53 (29.1%) of 182 tumors compared with 34 (18.3%) of 182 carcinomas by routine H-E staining (P = 0.001). D2-40 LVI was detectable in 81.0% (17/21) of nodal-positive tumors and significantly predicted lymph node metastasis (P = 0.001). Furthermore, D2-40 LVI was an independent prognostic factor for patients overall survival considering tumor stage, lymph node involvement, and tumor differentiation (P < 0.01). D2-40-negative tumors confined to the inner half of the myometrium showed an excellent outcome (5-year overall survival, 97.8%). D2-40-based LVI assessment improves the histopathological detection of lymphovascular invasion in endometrial cancer. Furthermore, LVI is of prognostic value and predicts lymph node metastasis. D2-40 LVI detection might help to select endometrial cancer patients who will benefit from a lymphadenectomy.

Impact of immunohistochemistry in sentinel lymph node biopsy in head and neck cancer

To determine the sensitivity, specificity, negative predictive value (NPV) and accuracy of hematoxylin-eosin (HE) staining compared to immunohistochemistry (IHC) in sentinel lymph node (SLN) histological analyses of head and neck squamous cell carcinoma. The Clinical prospective study was carried out at Tertiary referral university hospital. Patients with oral, lip and oropharyngeal squamous cell carcinoma undergoing elective neck dissection with clinically and radiologically negative necks were included. All patients were submitted to computer tomography scan for the evaluation of lymphatic metastases. The surgical procedure consisted of tumor resection, SLN sampling and elective neck dissection. Negative SLNs via HE were then submitted for IHC analysis of cytokeratin AE1/AE3 and step serial section (SSS). The main outcome measures were the negative predictive value of conventional HE staining techniques in the diagnosis of lymphatic metastases with the SSS/IHC analysis. Of 46 patients undergoing 63 neck procedures, 53 were SLN negative and 10 were positive on HE analysis. Using SSS/IHC analysis of these 53 negative SLNs on HE, two (3.8 %) were found to be positive. For HE, the sensitivity, specificity, NPV and accuracy were 77, 100, 94, and 95 %, respectively. With subsequent analysis with SSS/IHC, these values increased to 92, 100, 98 and 98 %, respectively. SSS/IHC is important in SLN analysis as the false negative rate decreased significantly while increasing the inherent sensitivity of the analyses.

Expression of bone morphogenetic protein 2, 4, 5, and 7 correlates with histological activity of otosclerotic foci

Conclusion: This study is the first to establish that bone morphogenetic protein 5 (BMP5) plays a role in the pathogenesis of otosclerosis. These results confirm that elevated expression levels of BMPs, members of the transforming growth factor (TGF)-β superfamily, contribute to the pathologically increased bone turnover in early, active stages of otosclerosis. Objectives: Otosclerosis is a complex bone remodeling disorder of the otic capsule, which might be characterized by increased expression of different types of BMPs. TGF-β and BMP are both members of the TGF-β superfamily and play a critical role in bone resorption and new bone formation. It has been suggested that BMP and its receptors may be involved in the pathologically increased bone turnover observed in otosclerosis. Methods: Fifty-one otosclerotic and 16 non-otosclerotic ankylotic stapes footplates were histologically analyzed: conventional hematoxylin-eosin staining and BMP2, 4, 5, and 7specific immunofluorescent assays were performed. Cortical bone fragments (n = 35) and incus specimens (n = 6) were used as negative controls. Results: Active otosclerosis (n = 39) was characterized by increased expression of BMP2, 4, 5, and 7. Inactive cases of otosclerosis (n = 12) were characterized by negative immunoreaction for BMPs. Non-otosclerotic stapes specimens (n = 16) and negative controls (n = 41) showed negligible BMP expression. The BMP expression pattern showed a strong correlation with the histological activity of otosclerosis.

Antihyperlipidemic Effect of Dietary Lentinus edodes on Plasma, Feces and Hepatic Tissues in Hypercholesterolemic Rats

We investigated diet supplementation with shiitake mushroom fruiting bodies on biochemical and histological changes in hypercholesterolemic rats. Six-wk old female Sprague-Dawley albino rats were divided into three groups of 10 rats each. A diet containing 5% Lentinus edodes fruiting bodies given to hypercholesterolemic rats reduced plasma total cholesterol, triglyceride, low-density lipoprotein (LDL), total lipid, phospholipids, and the LDL/high-density lipoprotein ratio by 34.33, 53.21, 75.00, 34.66, 25.73, and 71.43%, respectively. Feeding mushroom also significantly reduced body weight in hypercholesterolemic rats. However, it had no detrimental effects on plasma albumin, total bilirubin, direct bilirubin, creatinine, blood urea nitrogen, uric acid, glucose, total protein, calcium, sodium, potassium, chloride, inorganic phosphate, magnesium, or enzyme profiles. Feeding mushroom increased total lipid and cholesterol excretion in feces. The plasma lipoprotein fraction, separated by agarose gel electrophoresis, indicated that L. edodes significantly reduced plasma β and pre-β-lipoprotein but increased α-lipoprotein. A histological study of hepatic cells by conventional hematoxylin-eosin and oil red-O staining showed normal findings for mushroom-fed hypercholesterolemic rats. These results suggest that shiitake mushrooms could be recommended as a natural cholesterol lowering substance in the diet.