Paraffin Section Immunohistochemistry Research Articles

Getting two birds with one stone: Combining immunohistochemistry and Azan staining in animal morphology.

Classical histological stained sections have the disadvantage that fine structures, like individual neurites, or specific macromolecules, like neurotransmitters cannot be visualized. Due to its highly specific staining of only one target molecule within the cell, the visualization of delicate structures, which would be superimposed by other tissue layers in classical Azan staining, is possible with immunohistochemistry. However, using immunohistological methods not all tissues of a specimen can be visualized at once. In contrast, density specific stains like Azan allow for a whole staining of the tissues. We provide a step by step protocol of how to combine immunohistochemistry and Azan staining in the same serial paraffin sections. The combination of both methods allows for a highly detailed investigation of structures of interest. The spatial detection of the previous, to Azan staining, gained antibody-labeled signal allows for a much better understanding of animal organ systems. By using serial sections, it is possible to create an aligned image stack that is both Azan stained and also antibody-labeled. Thus enabling a correlative approach that bridges traditional histology with immunohistochemistry in animal morphology.

A new method for physical disector analyses of numbers and mean volumes of immunohistochemically labeled cells in paraffin sections

BackgroundIn the neurosciences, the physical disector method represents an established quantitative stereological method for unbiased sampling and counting of cells in histological tissue sections of known thickness. Physical disector analyses are conventionally performed using plastic-embedded tissue samples, because plastic-embedding causes a comparably low and definable shrinkage of the embedded tissue, and the thickness of thin plastic sections can be determined adequately. However, immunohistochemistry protocols often don’t work satisfactorily in sections of plastic-embedded tissue. New methodHere, a new methodological approach is presented, allowing for physical disector analyses of immunohistochemically labeled cells in paraffin sections. The embedding-related tissue shrinkage is standardized by using defined tissue sample volumes and paraffin volumes, and the extent of tissue shrinkage can be determined accurately from the sample volumes prior to and after embedding. Co-embedding of polyethylene section thickness standards together with the tissue samples allows the precise determination of individual paraffin section thicknesses by spectral reflectance measurements. Results and comparison with existing method(s)The applicability of the new method is demonstrated by physical disector analysis of immunohistochemically identified somatotroph cells in paraffin sections of porcine pituitary gland tissue. With consideration of individual shrinkage factors and section thicknesses, the cell numbers and mean volumes estimated in paraffin disector sections do not significantly differ from the results obtained by analyses of plastic-embedded pituitary tissue samples of the identical animals (2.4% average difference). ConclusionsThe featured method enables combination of paraffin section immunohistochemistry and physical disector analyses for unbiased quantitative stereological analyses of different cell types.

Morphological and immunohistochemical approaches to the diagnosis of Hashimoto thyroiditis and mucosa associated lymphoid tissue lymphomas: An audit.

Most cases of autoimmune hypothyroidism are due to Hashimoto thyroiditis (HT). It is sometimes difficult to distinguish HT from mucosa associated lymphoid tissue (MALT) lymphoma using cytology or histology alone. This has necessitated the use of immunohistochemistry and other molecular techniques. A descriptive study was undertaken to determine the prevalence of MALT lymphoma and other associations of HT using histopathological and immunohistochemical methods. For testing of kappa and lambda antibodies, paraffin sections were prepared for immunohistochemical staining using a peroxidase-antiperoxidase immune complex method. Immunostaining in HT, demonstrated a polyclonal lymphoid population, as evidenced by dual positivity for kappa and lambda staining cells, whereas MALT lymphoma revealed a monoclonal lymphoid population, with strong positivity for kappa immunostaining and lack of lambda light chain expression. The proportion of MALT lymphoma in surgically treated cases of HT in the present study was 3.5% (95% CI: 0%–8.3%). The common malignancy associated with HT was papillary carcinoma of the thyroid (8.8%). Among the benign conditions, nodular colloid goiter (7%) was more commonly associated. Although modern molecular techniques are available for the confirmation of lymphomas, they are more expensive, time consuming and are available only in a few centers. Strict morphological criteria can differentiate HT from MALT lymphoma, but in suspicious cases, paraffin section immunohistochemistry using light chain restriction can offer comparable and reliable results. Since papillary carcinoma and MALT lymphoma have been associated with HT, these patients require careful surveillance.

Actinin-4 splice variant - a complementary diagnostic and prognostic marker of pancreatic neuroendocrine neoplasms.

Introduction: For pathological diagnosis of pancreatic neuroendocrine neoplasms (pNENs) the routinely used immunohistochemical markers are chromogranin A (CgA) and synaptophysin (Syn). Their ability as prognostic markers is not well established. A splice variant of actinin-4 (Actn-4sv) was recently found to be an excellent biomarker of neuroendocrine neoplasms of the lung. We aimed to investigate the expression of Actn-4sv in pNENs and evaluate its quality as a biomarker of pNENs.Methods: Paraffin-embedded and frozen tissues specimens from 122 pNENs were analyzed. Western blots were performed to prove and compare the relative amount of Actn-4sv expression in pNENs tissue homogenates. For comparison pancreatic ductal adenocarcinoma (PDAC) and normal pancreatic tissues were analyzed in parallel. Immunohistochemistry (IHC) of paraffin sections of pNENs for Actn-4sv were performed and compared to the classic neuroendocrine markers CgA and Syn. Correlations were calculated between the staining intensity and distribution of Actn-4sv and staging, grading and afflicted lymph nodes respectively.Results: Actn-4sv was expressed in 88.5% (108/122) of pNENs, but not in normal pancreatic tissues (0/14) or PDAC (0/14). Compared to CgA and Syn, Actn-4sv was not detectable in islet cells of the normal pancreas. Staining intensity of Actn-4sv on pNENs negatively correlated to the histological grading (Spearman r=-0.4990, p<0.0001) and staging (r = -0.2581, p = 0.0041) but no correlation to afflicted lymph nodes was found. A significantly better overall survival was observed for pNEN patients with higher expression of Actn-4sv (hazard ratio 2.7; log-rank test p= 0.0349).Conclusions: The expression of Actn-4sv may be an important prognostic factor for patients with pNENs. Its expression correlates with the grading and staging of the tumors.

Relevance of host tau in tau seeding and spreading in tauopathies.

Human tau seeding and spreading occur following intracerebral inoculation of brain homogenates obtained from tauopathies in transgenic mice expressing natural or mutant tau, and in wild-type (WT) mice. The present study was geared to learning about the patterns of tau seeding, the cells involved and the characteristics of tau following intracerebral inoculation of homogenates from primary age-related tauopathy (PART: neuronal 4Rtau and 3Rtau), aging-related tau astrogliopathy (ARTAG: astrocytic 4Rtau) and globular glial tauopathy (GGT: 4Rtau with neuronal deposits and specific tau inclusions in astrocytes and oligodendrocytes). For this purpose, young and adult WT mice were inoculated unilaterally in the hippocampus or in the lateral corpus callosum with sarkosyl-insoluble fractions from PART, ARTAG and GGT cases, and were killed at variable periods of three to seven months. Brains were processed for immunohistochemistry in paraffin sections. Tau seeding occurred in the ipsilateral hippocampus and corpus callosum and spread to the septal nuclei, periventricular hypothalamus and contralateral corpus callosum, respectively. Tau deposits were mainly found in neurons, oligodendrocytes and threads; the deposits were diffuse or granular, composed of phosphorylated tau, tau with abnormal conformation and 3Rtau and 4Rtau independently of the type of tauopathy. Truncated tau at the aspartic acid 421 and ubiquitination were absent. Tau deposits had the characteristics of pre-tangles. A percentage of intracellular tau deposits co-localized with active (phosphorylated) tau kinases p38 and ERK 1/2. Present study shows that seeding and spreading of human tau into the brain of WT mice involves neurons and glial cells, mainly oligodendrocytes, thereby supporting the idea of a primary role of oligodendrogliopathy, together with neuronopathy, in the progression of tauopathies. In addition, it suggests that human tau inoculation modifies murine tau metabolism with the production and deposition of 3Rtau and 4Rtau, and by activation of specific tau kinases in affected cells.

Prognostic Impact of Morphology, MYC Gene Partner and BCL2/BCL6 Translocation Status in "High Grade B-Cell Lymphomas with MYC and BCL2 and/or BCL6 Rearrangements"

Introduction: "High grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements" (HG DH/THL) is a new diagnosis per the revised 2016 WHO classification. It has been colloquially called "double hit/triple hit lymphoma" and is significant for its aggressive clinical course. However, under this umbrella diagnosis there are several morphologic and cytogenetic variants with either controversial or unknown clinical significance. Herein, we investigate the prognostic significance of several of these parameters, including morphology, immunoglobulin gene (IG) vs non-IG MYC translocation partners, and BCL2/BCL6 translocation status in HG DH/THL in the Mayo Clinic Rochester practice.Materials and methods: All cases, which were identified through the Mayo Clinic Lymphoma Database, University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER), and the Mayo Clinic Cytogenetics Laboratory, had diffuse large B-cell lymphoma (DLBCL) or high grade B-cell lymphoma (HGBCL) morphology, were CD20-positive, and had a MYC translocation with concurrent BCL2 and/or BCL6 translocations. Morphology was re-reviewed by four hematopathologists when possible. Cell of origin (germinal center B-cell (GCB) vs non-GCB phenotype) was determined by paraffin section immunohistochemistry using the Hans classifier. Interphase FISH was performed on either tissue paraffin sections or bone marrow aspirate smears using a breakapart probe for MYC, followed by dual fusion probes for IGH/MYC, IGL/ MYC and IGK/MYC, a BCL6 breakapart probe, and either a BCL2 breakapart probe or an IGH/BCL2 dual fusion probe. Outcome analysis was restricted to patients with de-novo HG DH/THL or HG DH/THL at transformation of a previously diagnosed low-grade lymphoma and who were treated with anthracycline-based chemotherapy. Overall survival (OS) was defined as time from DH/THL diagnosis to death from any cause.Results: There were 100 patients with a median age of 61 years (range 29-87). The consensus re-review morphologic diagnosis (n=65) was HGBCL in 39 (60%) and DLBCL in 26 (40%). The HGBCL group had inferior survival (2-year OS = 35%) compared to DLBCL (2-year OS=62%, p= 0.058). By interphase FISH, the MYC translocation partner was an IG gene (IG/MYC) in 52 cases (60%; 39 IGH, 6 IGK and 7 IGL) and a non-IG gene (non-IG/MYC) in 35 cases (40%). No difference in OS was identified between IG/MYC and non-IG/MYC cases (p=0.85, Figure 1). By FISH, 87 cases (87%) had a BCL2 translocation (59 MYC/BCL2, 20 MYC/BCL2/BCL6, and 8 MYC/BCL2/[BCL6 unknown]) and 13 (13%) lacked a BCL2 translocation (MYC/BCL6). Those with a BCL2 translocation were all of GCB phenotype (N=85), while 6 of 12 of the MYC/BCL6 cases (50%) were of non-GCB phenotype (p <0.0001). All 13 MYC/BCL6 cases were de novo diagnoses, while 38% of cases with a BCL2 translocation represented either transformation (n=22) or recurrence (n=11) events (p=0.025). The MYC/BCL6 group had better survival (2-year OS=73%) than those with BCL2 translocation (2-year OS=43%, p= 0.13).Conclusion: As HG DH/THL's frequently have morphologic characteristics of DLBCL (40% in this series), it is important to perform complete interphase FISH analysis on all cases with DLBCL or HGBCL morphology to exclude the possibility of HG DH/THL. Furthermore, within the diagnosis of HG DH/THL, there are several morphologic and cytogenetic subtypes that are biologically and clinically significant. High grade morphology (HGBCL) tends to predict for a more aggressive clinical course independent of all other parameters (p= 0.058). Unlike a recent report (Blood 2015;126:2466), our data suggest MYC translocation partner (IG versus non-IG) may have no impact on overall survival (Figure 1). HG DH/THL's with a BCL2 translocation appear restricted to GCB phenotype, frequently represent either transformation or recurrence events, and tend to have inferior OS. In contrast, those without BCL2 translocations may be of either GCB or non-GCB phenotype, typically arise de novo, and tend to have a superior OS. Careful morphologic review and complete interphase FISH studies are essential in all HG DH/THL for prognostic and potential therapeutic purposes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Isocitrate dehydrogenase 2 (IDH2) is a mitochondrial NADP-dependent isocitrate dehydrogenase. It is considered to be a novel tumor suppressor in several types of tumors. However, the role and related mechanism of IDH2 in osteosarcoma remain unknown. The expression and significance of IDH2 were investigated by immunohistochemistry in formalin-fixed paraffin sections from 44 osteosarcoma patients. IDH2 was downregulated via lentiviral vector‑mediated RNA interference (RNAi) in the Saos-2 and MG-63 human osteosarcoma cell lines. The effect of IDH2 downregulation on human osteosarcoma was studied in vitro by MTT, flow cytometry and invasion assays. Nuclear factor-κB (NF-κB) and matrix metalloproteinase-9 (MMP-9) assays were also used to study the likely molecular mechanism of IDH2 downregulation on the malignant progression of osteosarcoma cells. The results revealed that the expression of IDH2 was inversely correlated with pathological grade and metastasis in osteosarcoma. IDH2 downregulation promoted a pro-proliferative effect on the Saos-2 and MG-63 osteosarcoma cell lines. IDH2 downregulation accelerated cell cycle progression from S to G2/M phase. The pro-proliferative effect induced by IDH2 downregulation may be ascribed to increased NF-κB activity via IκBα phosphorylation. The invasive activity of osteosarcoma cells was also significantly promoted by IDH2 downregulation and may result from elevated MMP-9 activity. In conclusion, IDH2 downregulation may exacerbate malignant progression via increased NF-κB and MMP-9 activity and may implicate the potential biological importance of IDH2 targeting in osteosarcoma cells. Downregulation of IDH2 exacerbates the malignant progression of osteosarcoma cells via increased NF-κB and MMP-9 activation.

Immunohistochemistry of Paraffin Sections from Mouse Ovaries.

Immunohistochemistry (IHC) is an efficient technique to detect cellular localizations of the proteins in paraffin-embedded tissues. It allows specific proteins to be visualized by the interaction of antibodies with an enzyme-substrate-chromogen system. Here, we describe indirect immunohistochemistry method for paraffin-embedded mouse ovaries fixed with Bouin's Fixative.

Proteomics and mass spectrometry in the diagnosis of renal amyloidosis.

The amyloidoses are a ‘group’ of disorders, all of which are associated with deposits that display similar staining and ultrastructural features and are toxic to tissues. Many proteins—currently 31 protein types and many more variants—have been shown to undergo such transformations. Among the various currently known amyloidoses, there are marked differences with regard to their pathogenesis and incidence, while the associated clinical picture is frequently overlapping. However, the therapies that are currently available are amyloid-type specific. The diagnosis of amyloidosis thus involves two steps: (i) a generic diagnosis, followed by (ii) an amyloid type-specific diagnosis or ‘amyloid typing’. Immunofluorescence in frozen sections or immunohistochemistry (IHC) in paraffin sections has traditionally been used in the typing of amyloid. However, IHC of amyloid differs significantly from IHC in other areas of surgical pathology; both caution and experience are necessary for its interpretation. The rationale for the application of proteomic methods to amyloid typing lies in the relative abundance of amyloid proteins in tissue where, frequently, it is the ‘dominant’ protein. Proteomic techniques include the following steps: sample preparation, protein extraction and digestion into peptide fragments, followed by their subsequent separation and measurement by mass spectrometry (MS) and protein identification by informatics. The advantages as well as the limitations of both methods—immunohistochemistry and MS-based proteomics—are discussed. The current recommendations for the application of proteomics in renal amyloidosis are summarized.

Exploratory development of rapid immunohistochemical staining technique in prostate biopsy tissue frozen section diagnosis

Objective To investigate the application value in the diagnosis of prostate biopsy tissue frozen section with rapid immunohistochemical detection of p40(ΔNp63),34βE12,p504s. Methods The expression of p40, 34βE12,p504s in 118 cases of prostate biopsy tissue frozen section detected by Max Vision rapid immunohistochemical staining technique. Conventional paraffin section immunohistochemistry of frozen remaining tissue served as a control. Results 118 cases of prostate biopsy histopathological diagnosis was divided into 79 cases of benign prostatic hyperplasia and 39 cases of prostate cancer. Max Vision rapid immunohistochemical staining technique was completed in 30minutes.Antibody expression located accurately. Background stained clearly and no interfering signal.The background was better than conventional immunohistochemistry of frozen remaining tissue. The positive expression rate of p40,34βE12,p504s with rapid immunohistochemistry in prostatic hyperplasia was 97.4%(77/79),93.7%(74/79), 0%(0/79),and in prostatic carcinoma was 0%(0/39),0%(0/39),97.4%(38/39).The positive expression rate of p40,34βE12,p504s with conventional immunohistochemistry in prostatic hyperplasia was 96.2%(76/79),93.7%(74/ 79),2.5%(2/79),and in prostatic carcinoma was 0%(0/39),0%(0/39),92.3%(36/39).The difference of expression between prostatic hyperplasia and prostatic carcinoma with rapid immunohistochemical detection p40 group:χ2= 109.402,P=0.000,34βE12 group:χ2=97.971,P=0.000,p504s group:χ2=113.537,P=0.000;The difference of expression between prostatic hyperplasia and prostatic carcinoma with conventional immunohistochemical detection p40 group:χ2=105.410,P=0.000,34βE12 group:χ2=97.971,P=0.000,p504s group:χ2=96.388,P=0.000;The expression of prostatic hyperplasia with between rapid immunohistochemical detection and conventional immunohistochemical detection 34βE12 group was identical,p40 group:χ2=0.207,P=0.649,p504s group:χ2=2.026,P=0.155; The expression of conventional immunohistochemical detection with between rapid immunohistochemical detection and conventional immunohisto-chemical detection p40 group and 34βE12 group were identical,p504s group:χ2=1.054, P=0.305.The expression of three markers between prostatic hyperplasia and prostatic carcinoma had statistical significance(P 0.05). Conclusion Max Vision rapid immunohisto-chemical staining technique has the advantages of rapid, accurate, timely. It could be used to rapid diagnosis of prostate biopsy tissue.The combined detection of p40,34βE12,p504s has very high practical value in the differential diagnosis of benign and malignant lesions of the prostate. Key words: Rapid immunohistochemistry; Prostate; p40; 34βE12; p504s; Diagnosis

Association of BCSC-1 with human esophageal squamous cell carcinoma.

Breast cancer suppressor candidate-1 (BCSC-1) is anewly identified candidate tumor suppressor gene. BCSC-1 shows decreased levels in a variety of cancer types. In this study, we investigated the association between BCSC-1 and human esophageal squamous cell carcinoma (ESCC). BCSC-1 expression was detected in ESCC and normal tissues adjacent to tumor tissues by Western blot analysis and real-time PCR as well as immunohistochemistry of paraffin sections. The relationships between BCSC-1 expression and various clinicopathological characteristics were analyzed. Western blot analysis and real-time PCR showed that levels of BCSC-1 protein and mRNA expression in ESCC significantly decreased compared with those in adjacent normal tissues. Immunohistochemistry exhibited marked reduction of BCSC-1 in 38 of 105 ESCC specimens. Moreover, downregulation of BCSC-1 was associated with the grade of tumor cellular differentiation (P<0.05). These findings indicate that BCSC-1 downregulation in ESCC is associated with carcinogenesis and may play important roles during the process of ESCC cancer development.

Expression of TGFβ-1 and EHD1 correlated with survival of non-small cell lung cancer.

Transforming growth factor-β1 (TGFβ-1) signaling is regulated by endocytotic pathway. To clarify the prognostic value of TGFβ-1 and to verify the involvement of endocytosis in drug resistance, we examined the expression of TGFβ-1 and Eps15 homology domain 1 (EHD1) in non-small cell lung cancer (NSCLC) and its association with tumor characteristics and survival of patients with NSCLC. Expression of TGFβ-1 and EHD1 was evaluated by immunohistochemistry in paraffin sections from 105 NSCLC patients. Overall survival (OS) was analyzed by Kaplan-Meier method, log-rank test, and multivariate Cox proportional hazard regression model. Positive immunostaining of TGFβ-1 and EHD1 was detected in 52.38 and 39.05 % of NSCLC samples, respectively. In non-adjuvant chemotherapy-treated group (P = 0.006) and epidermal growth factor receptor (EGFR) (+) group (P = 0.038), patients with TGFβ-1 expression had a longer OS. EHD1 negative expression predicted a longer OS (P = 0.003), especially in EGFR (+) (P = 0.006) and adjuvant chemotherapy-treated patients (P = 0.003). NSCLC patients with concurrent positive TGFβ-1 and negative EHD1 (combined markers) were significantly correlated with better OS (P = 0.001). American Joint Committee on Cancer (AJCC) status and combined markers were independent prognostic indicators for OS (HR (95 % CI) 1.576 (1.112-2.232), P = 0.011 and HR 0.349 (0.180-0.673), P = 0.002, respectively). We identified concordant TGFβ-1 positive and EHD1 negative as a strong favorable prognosis factor in NSCLC. Our results may help us to select and optimize strategies for individualized therapy.

Constitutive Expression of HCA2 in Human Retina and Primary Human Retinal Pigment Epithelial Cells

Purpose/Aim: HCA2, a receptor of β-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells.Materials and methods: Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis.Results: Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells.Conclusions: Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target.

Increased EHD1 in non-small cell lung cancer predicts poor survival.

One of the main challenges of lung cancer research is identifying patients at high risk for recurrence after surgical resection. We evaluated the prognostic power of four proteins in the endocytic pathway in 114 non-small cell lung cancer patients (NSCLC). We tested the four proteins (epidermal growth factor receptor [EGFR], RAB11FIP3, EHD1, and caveolin-1), critical nodes in the endocytosis/recycling pathway, by immunohistochemistry in paraffin sections from 114 non-small cell lung cancer patients. We analyzed the correlation between our target proteins and clinical variables. Within these variables, an overall survival (OS) prediction model was constructed using Cox proportional hazard regression. EHD1 expression correlated with gender (P = 0.001), histology type (P < 0.001), and EGFR expression (P = 0.008), but not with any of the other clinical parameters. Statistical correlation analysis showed that the expression of EHD1 positively correlated with high level of EGFR (P < 0.001) and RAB11FIP3 (P < 0.001), and the expression of caveolin-1 positively correlated with high level of EGFR (P < 0.001) in the NSCLC samples. EHD1 expression was an OS prognostic factor for all of the patients (P = 0.009), for the group of adjuvant chemotherapy-treated patients (P = 0.006), and for the EGFR positive patients (P = 0.034). We identified EHD1 as a strong prognostic predictive factor in NSCLC. The expression level of EHD1 would potentially be useful in developing customized strategies for managing lung cancer, such as the selection of patients eligible for chemotherapy.

Low ING4 protein expression detected by paraffin-section immunohistochemistry is associated with poor prognosis in untreated patients with gastrointestinal stromal tumors

Inhibitor of growth 4 (ING4) has deserved attention as a tumor suppressor gene in many malignant tumors. In our study, we investigated ING4 immunoexpression in gastrointestinal stromal tumors (GISTs) and its prognostic value. The expression of ING4 and Ki67 was investigated in 41 samples of various risk gastrointestinal stromal tumors by immunohistochemical technique. The associations of ING4 expression and clinicopathological parameters, and prognosis of the patients, were analyzed by multivariate Cox regression analysis. ING4 expression showed a decreased trend from lower-risk to high-risk gastrointestinal stromal tumors, and an opposite trend for Ki67 expression. In lower-risk tumors, it was found the expression level of ING4 was 78.95 % ± 27.90 % and that of Ki67 was 4.42 % ± 3.75 %. However, in high-risk tumors, the expression level of ING4 was 9.23 % ± 7.66 % and that of Ki67 was 18.50 % ± 9.09 %. There was a strongly negative correlation between the expression levels of ING4 and Ki67. A significant difference was observed in the expression of ING4 between invasion and non-invasion (p < 0.001). The expression of ING4 was markedly correlated with tumor size (p < 0.001), mitotic index (p < 0.001), tumor necrosis (p = 0.021), invasion (p < 0.001), recurrence and metastasis (p = 0.021), and mortality (p < 0.001). The low expression level of ING4 protein was correlated with high-risk GISTs. ING4 might be involved in the progression of GISTs and inhibit its invasion. ING4 might be one of the prognostic indicators in GISTs.

Lymphoma classification and the tools of our trade: an introduction to the 2012 USCAP Long Course

The 2012 USCAP Long Course ‘Malignant Lymphomas—Building on the Past, Moving to the Future’ began with an introduction to lymphoma classification over the last half century and a discussion of our current diagnostic armamentarium, together with a look toward the future. The Rappaport classification, originally published in 1956, was a morphologic classification with few categories. The early 1970s saw a great and tumultuous revolution in the field with the publication of two functional lymphoma classifications that related the malignant lymphomas to the cells of the normal immune system—the Lukes/Collins classification from the United States and the Kiel classification from Professor Lennert and the European Lymphoma Club. With discord abounding, the NCI working formulation, published in 1982, satisfied some but was a step back to a morphologic-based classification. In 1994, the International Lymphoma Study Group published the REAL classification, which reflected state-of-the-art practice for that time, and was shortly followed by preparations for the modern World Health Organization (WHO) classification published in 2001 and revised in 2008. The WHO classification, created by hematopathologists working with the advice and consent of clinical hematologist/oncologists, recognizes numerous distinct entities, defined based on their histopathological, immunophenotypical, molecular/cytogenetic and clinical features. The classification requires use of a multiparameter approach to lymphoma diagnosis although we still rely heavily on histopathology. Immunophenotypical studies, whether using paraffin section immunohistochemistry and/or flow cytometry, are also critical in almost all circumstances. Molecular/cytogenetic techniques that are constantly changing have an increasingly important role, even if not always required. The full impact of next-generation sequencing is yet to be felt but we are beginning to catch a glimpse of what is in our future. Finally, one must not forget the great importance of clinical data in arriving at a diagnosis that best serves the patient, our ultimate goal.

Untersuchungen des maternalen und fetalen Serumlipidprofils und des oxidierten ‚Low Density‘ Lipoproteins im plazentaren Gewebe bei Präeklampsie und normotensiven Schwangerschaften

Oxidised low density lipoproteins (oxLDL) are key players in the development of atherosclerotic cardiovascular diseases. Since there are similarities between the pathogenesis of preeclampsia and atherosclerosis we hypothesised an increased accumulation of oxLDL at the materno-foetal and foeto-foetal interface within the placental tissue of preeclamptic women compared to women with normotensive pregnancies (controls). Moreover, we analysed maternal and foetal serum lipid parameters. oxLDL was determined by immunohistochemistry in placental paraffin sections of 14 women suffering from preeclampsia (30th-39th week of gestation) and compared to 28 preterm and term deliveries (25th-40th week of gestation). 10 high power fields were chosen randomly by the newCAST software and oxLDL expression was analysed via standardised methods by 2 independent and blinded investigators. Maternal and foetal triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol were measured. Statistical examination was carried out by the Mann-Whitney test. oxLDL was found in villous trophoblast and placental endothelium. No significant differences were observed in expression intensity between preeclampsia and controls. Maternal and foetal triglyceride levels were significantly increased in preeclampsia compared to controls (pre-eclampsia mothers: 293 [SD 87.4] mg/dL, controls: 214 [SD 89.4] mg/dL, p=0.0097; preeclampsia foetuses: 26 [SD 16.6] mg/dL, controls: 18 [SD 10.4] mg/dL, p=0.0463). No significant differences in other lipid concentrations were found. We could not confirm our initial hypothesis of an increased oxLDL accumulation in placental tissue of preeclampsia. However, preeclampsia is a condition of dyslipidaemia affecting both maternal and foetal serum with implications for development and programming of cardiovascular diseases in later life.

Dyskerin Overexpression in Human Hepatocellular Carcinoma Is Associated with Advanced Clinical Stage and Poor Patient Prognosis

BackgroundDyskerin (encoded by the DKC1 gene) is an essential nucleolar protein involved in cell proliferation, where it is required for the pseudo-uridylation of ribosomal RNA (rRNA) molecules and the stabilization of the telomerase RNA component. Dyskerin expression has been reported to predict poor survival in some cancer patients. The aim of the present study was to analyze the expression of dyskerin in hepatocellular carcinoma (HCC) and to determine its correlation with clinicopathologic features, including the survival of patients with HCC.Methodology/Principal FindingsDyskerin protein expression was detected by immunohistochemistry in paraffin sections of 252 HCC cases and 80 noncancerous liver tissues. The correlation was analyzed between dyskerin expression levels and clinicopathologic variables and prognosis. Dyskerin protein was significantly overexpressed in HCC tissues when compared to noncancerous liver tissue. Dyskerin overexpression was positively correlated with the hepatitis B surface antigen status, serum alpha-fetoprotein, and advanced clinical stage in HCC patients. A survival analysis indicated that HCC patients with higher dyskerin expression had a significantly shorter overall survival and 5-year survival time when compared to those with low expression. A multivariate analysis suggested that dyskerin overexpression was an independent factor for prognosis (hazard risk, 2.912; P = 0.007). Expression of DKC1 mRNA was measured by quantitative RT-PCR in 80 HCC and 50 non-cancerous tissues. The relationship between DKC1, TERT, MKI67, and MYC mRNA expression in HCC tissues was also evaluated. DKC1 mRNA was significantly overexpressed in HCC tissues and showed a significant correlation with MKI67 and MYC mRNA but a weak correlation with TERT mRNA.Conclusions/SignificanceDyskerin overexpression in HCC patients was correlated with MYC and MKI67 expression and showed a possible involvement in the tumorigenic process. Dyskerin overexpression may be an unfavorable prognostic factor in patients with HCC.

PP013. Oxidized low density lipoprotein accumulation and the expression of the lectin-like oxLDL receptor (LOX-1) in placental tissue in preeclampsia and healthy controls

Oxidized Low Density Lipoproteins (oxLDL) and its receptor the lectin-like oxLDL receptor 1 (LOX-1) are key players in the development of atherosclerotic cardiovascular diseases. Since preeclampsia is known to share similarities to the pathogenesis of atherosclerosis we hypothesized an increased accumulation of oxLDL and an increased expression of LOX-1 at the materno-fetal and feto-fetal interface within the placental tissue in preeclampsia in comparison to a control group. Second, we analyzed maternal and fetal serum lipid parameters including fetal oxLDL concentration. OxLDL and LOX-1 intensity was determined via immunohistochemistry in placental paraffin sections of 11 women suffering from preeclampsia and compared to 11 gestational age matched preterm deliveries (29th to 36th week of gestation). Ten 'High Power Fields' were chosen randomly by the newCAST software and expression was analyzed via standardized methods by two independent and blinded observers. Maternal and fetal triglycerides, total cholesterol, LDL-cholesterol and HDL-cholesterol were measured by enzymatic colorimetric methods. Fetal oxLDL serum concentration was estimated by ELISA. Statistical examination was carried out by Student's t-test. Skewed variables were log-transformed. oxLDL and LOX-1 was predominantly found to be in villous trophoblast and placental endothelium. No significant differences could be observed in oxLDL expression intensities between preeclampsia and controls. LOX-1 expression tended to be increased in placental trophoblast and endothelium without being statistical significant (Table 1). Fetal triglyceride levels were significantly elevated in preeclampsia compared to controls while maternal triglyceride levels tend to be increased. No other significant differences in lipid concentrations could be observed (Table 2). We could not confirm our initial hypothesis of an accelerated oxLDL accumulation in placental tissue of preeclampsia. Though not statistically significant, placental endothelium seems to be activated in preeclampsia since LOX-1 expression is increased. Moreover, preeclampsia is a condition of dyslipidemia affecting both, maternal and fetal serum with implications for development of cardiovascular diseases in later life.

PP014. Estimating fully and minimal oxidized low density lipoprotein accumulation in placental tissue in intrauterine growth restriction and healthy controls

We recently demonstrated that maternal serum LDL- and fetal serum HDL-cholesterol concentration is significantly reduced in intrauterine growth restriction (IUGR) [1]. We now hypothesized that increased oxidative stress in IUGR placenta leads to an accumulation of oxidized LDL (oxLDL) particles which then become trapped within the placenta subsequently leading to reduced availability of cholesterol for mother and fetus. Fully oxidized LDL (oxLDL) was determined via immunohistochemistry in placental paraffin sections of 18 women suffering from IUGR and 18 gestational age matched controls. Ten 'High Power Fields' were chosen randomly by the newCAST software and oxLDL expression was estimated via standardized methods by two independent and blinded observers. Minimal oxidatively modified LDL (MM-LDL) and non-modified Apolipoprotein B (ApoB) concentration was measured in full placental tissue lysates by ELISA. Values were correlated with maternal and fetal total cholesterol, LDL-, and HDL-cholesterol concentrations. Statistical examinations were carried out by Student's t-test and calculation of Pearson's correlation coefficient. oxLDL was found predominantly to be in villous trophoblast and placental endothelium. OxLDL intensity tended to be increased in IUGR (Table 1). We found MM-LDL concentrations in whole placental tissue lysates to be highly correlated to placental ApoB concentration (r=0.93). Both parameters were non-significantly decreased in placenta of IUGR compared to controls (Table 1). Maternal serum LDL-C, and fetal serum LDL-C, TC, and HDL-C concentrations were significantly decreased in IUGR compared to controls (Table 2). OxLDL staining intensity was mildly negatively correlated to maternal LDL-C (r=-0.315) and much less to fetal HDL-C concentrations (r=-0.212). Placental ApoB and MM-LDL concentration were moderately positively correlated with fetal HDL-C concentrations (r=0.492 and r=0.447). Conformational changes of the ApoB lipoprotein during the process of oxidation might lead to an accumulation of oxLDL particles in placental tissue of IUGR and reduced fetal cholesterol bioavailability as evidenced by a decrease in fetal serum cholesterol levels. However, our analysis lacks in sufficient power and further studies are underway focussing on that subject.