Presence Of Staining Research Articles

Ber-EP4 staining patterns on basal cell carcinomas.

This article aimed to study two different parameters of basal cell carcinoma (BCC): First, to analyze the expression of antihuman epithelial antigen (Ber-EP4) on the primary and recurrent BCCs on the head, neck, and other body parts and second, to find Ber-EP4's staining pattern and staining intensities correlation between histological type, demographic data, tumor, and its prognostic parameters. We evaluated the Ber-EP4 staining patterns of 201 patients diagnosed with BCC. We analyzed the possible correlation between the tumor's prognostic parameters and the Ber-EP4 staining intensity and its pattern (peripheral, superficial, or diffused). In 199 out of the 201 cases, staining was observed. Two cases were unstained. In 25.6% (n=51) of the cases with staining, the staining was weak, on the 25.6% (n=51), it was moderate, and on the 48.8% (n=97), it was severe. The staining pattern was 31.2% (n=62) peripheral, 4.0% (n=8) superficial, 54.7% (n=109) diffuse, and 10.1% (n=20) peripheral and superficial. Ber-EP4 is the only antibody commonly used for BCC diagnosis; the existence of different staining intensities and patterns in BCC tumor cells in routine dermatopathology practice limit the pathologists. The studies investigating Ber-EP4 staining in BCCs were conducted with very small numbers of cases. In these studies, even the presence of staining in the focal area was considered to be a positive acceptance criterion; the staining intensity and pattern were not evaluated. Therefore, our study is the first study with a high number of cases and the first to include an evaluation of Ber-EP4 staining's intensity and localization.

Canadian ROS proto-oncogene 1 study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories.Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor.Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases.This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

Correlation of p16 immunostaining in cell-blocks with corresponding tissue specimens for squamous cell carcinomas of the oropharynx.

The goal of this study was to evaluate the performance of p16 staining in cell-blocks vs tissue specimens as a surrogate marker for human papillomavirus (HPV) status in oropharyngeal squamous cell carcinomas. Head and neck squamous cell carcinoma cases presenting as a neck mass with a p16 result on cytology and corresponding tissue specimens (1 January 2014 to 30 June 1920) were included in the study. The following were assessed from cell-block material: number of tumour clusters, percentage of tumour cells with p16 staining, and presence of staining in clusters vs single cells. Results were compared to tissue p16 status. Results of any other ancillary HPV testing were also noted. Forty-two head and neck squamous cell carcinoma neck metastases (35 oropharyngeal, five non-oropharyngeal, and 2 unknown primaries) were identified. The p16 staining pattern in cell-blocks was seen in single cells (27.6%), clusters (44.8%), or both (27.6%). The percentage of tumour cells staining for p16 in cell-blocks was much lower than in corresponding tissue specimens. There were four false negatives and one false positive (concurrent HPV DNA polymerase chain reaction testing was positive in cytology and surgical material). Compared to tissue, the cut-off for p16 interpretation in cell-blocks is substantially lower and staining may be present in single cells or clusters. In 96.9% of cases, any p16 staining in cell-blocks correlated with positive p16 staining in surgical specimens. However, a negative or discrepant p16 result on cell-block should prompt confirmatory HPV studies, as false negative p16 staining in cell-blocks is high.

Clinical and laboratory evaluation of the effects of different treatment modalities on titanium healing caps: a randomized, controlled clinical trial

The objective of this study is to evaluate the effects of treatment modalities on titanium surface characteristics and surrounding tissues. Eighteen participants each had four titanium healing caps (HC) attached to four newly inserted implants. After healing, each HC was randomly assigned to either (1) titanium curettes (TC), (2) stainless steel ultrasonic tip (PS), (3) erythritol air-polishing powder (EP), or (4) only rubber cup polishing (CON). Probing depths (PD), bleeding on probing (BOP), matrix metalloproteinase 8 (MMP-8), and periopathogens were recorded before and 3months following instrumentation. After final assessments, HCs were removed, cleaned, and subjected to (a) bacterial colonization (Streptococcus gordonii, 24h; mixed culture, 24h) and (b) gingival fibroblasts (5days). HC surfaces were analyzed with a scanning electron microscope (SEM). No significant differences between the groups were evident before or after instrumentation for PD and BOP (except TC showed a significant decrease in PD; p = 0.049). MMP-8 levels and bacterial loads were always very low. MMP-8 decreased further after instrumentation, while bacteria levels showed no change. No significant differences (p > 0.05) were evident in bacterial colonization or fibroblast attachment. A comparison of the overall mean SEM surface roughness scores showed a significant difference between all groups (p < 0.0001) with the lowest roughness after EP. All treatments performed yielded comparable outcomes and may be implemented safely. Clinicians may fear implant surface damage, but all instrumentation types are safe and non-damaging. They can be implemented as needed upon considering the presence of staining and soft and hard deposits.

Value of detection of DNA mismatch repair proteins deficiency by immunohistochemistry in predicting tumor microsatellite status

To evaluate the sensitivity and specificity of immunohistochemical (IHC) staining of DNA mismatch repair (MMR) protein for the screening of microsatellite instability (MSI) colorectal cancer (CRC). A total of 255 CRC cases were studied, including 140 cases of routine paraffin-embedded tissue samples and 115 cases constructed on tissue microarray. Expressions of 4 MMR proteins including MHL1, MSH2, MSH6 and PMS2 were investigated by IHC. Negative protein expression was defined as complete absence of nuclear staining within tumor cells in the presence of positively labeled internal non-neoplastic cells. Focal staining was defined as the presence of staining in < 5% of the tumor cells. CRCs showing negative staining for any MMR proteins were interpreted as MMR deficient tumors. PCR-genescan MSI analysis was performed in each case by a five marker panel including Bat26, Bat25, NR-21, NR-24 and MONO-27. Among the 140 CRCs with routine formalin-fixed paraffin embedded tissue sections, concordance rate between IHC and PCR-genescan was 98.6% (138/140), the sensitivity and specificity of IHC in detecting MSI tumors were 94.9% (37/39) and 100.0% (101/101), respectively. The 2 disconcordant cases showed focal staining in at least one of the MMR proteins but were confirmed to be MSI-H CRCs by PCR-genescan assay. On tissue microarray, 91.3% (105/115) of the cases had informative results. The concordance rate between IHC and PCR-genescan was 100.0% (105/105). Both the specificity and sensitivity of IHC in detecting MSI tumors on available tissue microarray samples were 100.0%. Ten cases were inclusive due to the presence of negative stains of MMR proteins in both the tumor and internal control cells. Detection of 4 MMR proteins expression by IHC is reliable for identifying MSI CRCs and is recommended for routine practice. Tumors with focal MMR protein staining are highly suspected for the presence of MSI-H and PCR-genescan based MSI analysis should be performed to confirm.

SONODYNAMIC EFFECT OF EMODIN ON MACROPHAGES

ObjectivesEmodin has been used as an anti-inflammatory agent, and inflammation is a crucial feature of atherosclerosis. Here, we investigated the sonodynamic effect of emodin on macrophages, the pivotal inflammatory cells...

MUC2 Is a Highly Specific Marker of Goblet Cell Metaplasia in the Distal Esophagus and Gastroesophageal Junction

Currently, the American College of Gastroenterology requires identification of goblet cells in mucosal biopsies from the esophagus to diagnose Barrett esophagus (BE). Identification of goblet cells in mucosal biopsies is fraught with limitations such as sampling and interpretation error. One previous study by our group suggested that MUC2 expression in esophageal nongoblet columnar cells represents a late biochemical reaction in the conversion of mucinous columnar cells to goblet cells in BE. We conducted this study to evaluate the prevalence, sensitivity, and specificity of MUC2 positivity in nongoblet columnar epithelium for detection of goblet cells in the distal esophagus and gastroesophageal junction (GEJ) region. We also sought to identify associations between MUC2 positivity and clinical and endoscopic risk factors for BE. This analysis utilized mucosal biopsies of the distal esophagus or GEJ from 100 patients who participated in a community clinic-based study of patients with chronic gastroesophageal reflux disease evaluated prospectively in the western part of Washington state. We randomly selected 50 patients who had columnar epithelium with goblet cells, representing the study group and 50 patients without goblet cells, representing the comparison group. Immunohistochemistry for MUC2 was performed on samples in a blinded manner without knowledge of the clinical or endoscopic features of the patients. The presence of staining was noted in both goblet and nongoblet epithelium, both close to and distant from the mucosa with goblet cells, when the latter were present. All study patients showed MUC2 positivity in goblet cells. MUC2 was present in nongoblet columnar epithelium in 78% of study patients with goblet cells, but in only 4% of controls without goblet cells (P<0.0001) (sensitivity, 78%; specificity, 96% for goblet cell metaplasia). MUC2 was significantly more common in nongoblet columnar cells close to, rather than distant from, the mucosa with goblet cells (P<0.00001). Finally, MUC2 was significantly associated with endoscopic evidence of columnar metaplasia in the distal esophagus, and with known risk factors for BE, such as older age, white race, frequent heartburn, and elevated body mass index. We conclude that goblet cells likely develop from a field of MUC2-positive mucinous columnar cells, and as such, MUC2 represents a late event in the development of goblet cells. MUC2 staining in nongoblet columnar cells is a reasonably sensitive and highly specific marker for goblet cells in the distal esophagus and GEJ, and its presence is predictive of endoscopic columnar metaplasia of the esophagus, even in patients without goblet cells.

Irs2 Inactivation Suppresses Tumor Progression in Pten+/− Mice

Mutations in the phosphatase and tensin homologue (PTEN)/phosphatidylinositol-3 kinase-alpha (PI3K) signaling pathway are frequently found in human cancer. In addition, Pten(+/-) mice develop tumors in multiple organs because of the activation of the PI3K signaling cascade. Because activation of PI3K signaling leads to feedback inhibition of insulin receptor substrate-2 (IRS2) expression, an upstream activator of PI3K, we therefore anticipated that IRS2 expression would be low in tumors that lack PTEN. Surprisingly, however, an elevation of IRS2 was often detected in tumor samples in which PTEN levels were compromised. To determine the potential contribution of Irs2 to tumor progression, Pten(+/-) mice were crossed with Irs2(+/-) mice. Deletion of Irs2 did not affect the initiation of neoplasia found in Pten(+/-) mice but suppressed cancer cell growth, proliferation, and invasion through the basement membrane. Deletion of Irs2 also attenuated the expression of Myc in prostatic intraepithelial neoplasia in Pten(+/-) mice. In addition, the expression levels of IRS2 and MYC were highly correlated in human prostate cancer, and IRS2 could stimulate MYC expression in cultured cells. Our findings provide evidence that the PI3K-activating adaptor Irs2 contributes to tumor progression in Pten(+/-) mice by stimulating both Myc and DNA synthesis.

Increased cytoplasmic localization of KLF5 in prostate cancer

14652 Background: KLF undergoes genomic deletion, transcriptional downregulation, and excessive protein degradation in prostate cancer cells, but its protein expression has not been examined in prostate cancer specimens. Methods: We performed immunohistochemical staining on a tissue microarray composed of 37 benign prostate tissues, 58 primary prostate cancers, and 9 metastatic tumors. Cultured cells without KLF5 expression and those expressing KLF5 through gene transfection were used as controls. Each sample was scored for presence of staining, nuclear or cytoplasmic localization of staining, and intensity of staining (0, no staining; 1, below counterstain; 2, equal to counterstain; 3, strong staining obscuring counterstain). KLF5 expression was correlated with pathologic characteristics of tumors by performing Chi-square analysis and two tailed t tests. Results: KLF5 staining was noted in all 104 specimens, either as nuclear staining, cytoplasmic staining, or both. While no significant differences were seen in the intensity of the nuclear staining between benign and tumor groups, a significant increase in cytoplasmic staining was seen in tumor specimens (2.24 vs. 1.71, p = 0.0002). In addition, samples with high cytoplasmic and low nuclear KLF5 were more likely to be tumor samples (p = 0.0413). No significant differences were seen between samples with Gleason grades, or between primary tumors and metastases. Conclusions: Increased cytoplasmic localization of KLF5 may impair KLF5’s transcription factor function in the development and/or progression of prostate cancer. No significant financial relationships to disclose.

Gangliosides Inhibit Flagellin Signaling in the Absence of an Effect on Flagellin Binding to Toll-like Receptor 5

A recent study (Ogushi, K., Wada, A., Niidome, T., Okuda, T., Llanes, R., Nakayama, M., Nishi, Y., Kurazono, H., Smith, K. D., Aderem, A., Moss, J., and Hirayama, T. (2004) J. Biol. Chem. 279, 12213-12219) concluded that gangliosides serve as co-receptors for flagellin signaling via toll-like receptor 5 (TLR5). In view of several findings in this study that were inconsistent with a role for gangliosides as co-receptors, we re-examined this important issue. Using TLR5-negative RAW 264.7 cells and a TLR5-enhanced yellow fluorescent protein chimera, we established an assay for specific binding of flagellin to cells. Inhibition of clatherin-mediated internalization of flagellin.TLR5-enhanced yellow fluorescent protein complexes did not impair flagellin activation of IRAK-1. Thus flagellin signal occurs at the cell surface and not intracellularly. Exogenous addition of mixed gangliosides (GM1, GD1a, and GT1b) as well as GD1a itself inhibited flagellin-induced interleukin-1 receptor-associated kinase activation as well as tumor necrosis factor alpha production in HeNC2, THP-1, and RAW 264.7 cells. Gangliosides inhibited flagellin signaling in the absence of an effect on flagellin binding to TLR5. Depletion of gangliosides in RAW 264.7 cells did not alter the concentration dependence or magnitude of flagellin signaling as measured by interleukin-1 receptor-associated kinase activation or tumor necrosis factor alpha production. Our findings are consistent with the conclusions that gangliosides are not essential co-receptors for flagellin and that the inhibitory effect of gangliosides is mediated by at least one mechanism that is distinct from any effect on the binding of flagellin to TLR5.

ID protein expression in oral epithelial dysplasia and squamous cell carcinoma

Id proteins were recently described as antagonizing basic helix-loop-helix transcription factors, which ordinarily play a key role in cell development, proliferation, and differentiation. Thus, Id overexpression correlates with cell proliferation and arrested differentiation. Id expression has been observed in normal skin and overexpression in a variety of carcinomas, but no studies have examined its expression in the oral cavity. To evaluate the possible role of Id in oral carcinogenesis, we examined immunohistochemical staining patterns for Id-1, Id-2, and Id-3 on sections of “normal” oral mucosa (n = 5) and sections with mild/moderate dysplasia (n = 5), severe dysplasia/carcinoma in situ (n = 5), well/moderately differentiated squamous cell carcinoma (SCC; n = 5) and poorly differentiated SCC (n = 5). The presence of staining was evaluated in basal/parabasal (B/PB), lower spinous (LS), upper spinous (US), and stratum corneum epithelial layers. Staining was considered focal if <50% and diffuse if >50% of cells stained. Staining intensity was graded on a scale of 0 to 3. Id-1 staining was cytoplasmic; in all 5 cases of normal epithelium, it was diffuse in the B/PB layer (average intensity [AI] = 2). Two cases of normal epithelium also exhibited focal LS staining (AI = 0.8). Among cases of mild/moderate dysplasia, all had diffuse B/PB (AI = 2.6), 3 diffuse and 2 focal LS (AI = 3), and 2 focal US staining (AI = 0.4). Among cases of severe dysplasia/carcinoma in situ, all 5 had diffuse B/PB and LS staining and 4 exhibited diffuse US staining (AI = 2.4, 2.2, and 1.6). No trends were seen for Id-2 and Id-3, which stained all but the stratum corneum in both normal and dysplastic epithelium. In 7 of 10 cases of oral SCC, Id-1, Id-2, and Id-3 stained more than 67% of tumor cells, with an AI range of 1.75 to 2.80. Id-1 expression generally increases in upper levels of oral epithelium as the degree of dysplasia increases, and Id-1, Id-2, and Id-3 overexpression is seen in oral SCC. Future studies will examine Id expression as a potential prognostic indicator in oral dysplasia and SCC.

Differential expression of CD44s and CD44v3-10 in adenocarcinoma cells and reactive mesothelial cells in effusions.

The detection of malignant cells in serous effusions obtained from patients diagnosed with cancer marks the presence of metastatic disease and is associated with a poor outcome. The purpose of this study was to evaluate the role of CD44s and CD44v isoforms in the distinction between mesothelial cells and malignant epithelial cells in effusions. Fifty-nine fresh pleural and peritoneal effusions were studied. These consisted of 41 specimens from patients with known gynecological neoplasms, 9 from patients diagnosed with breast adenocarcinoma, and 9 effusions from patients with various nongynecological malignancies or tumors of unknown origin. Forty-three effusions contained malignant/atypical epithelial cells, and 16 effusions were diagnosed as reactive. Three effusions contained exclusively malignant cells. Specimens were stained with anti-CD44s, v3, v5, v6, v7 and v3-10. The presence of staining in cancer cells, benign mesothelial cells and lymphocytes was evaluated. CD44s immunoreactivity was seen in 10 of 43 (23%) cases in malignant/atypical epithelial cells and in 53 of 56 (94%) cases in benign cells. In contrast, CD44v3-10 was seen in 23 of 43 (55%) cases in malignant/atypical epithelial cells and in 3 of 56 (6%) cases in benign cells. We advocate the use of CD44s and CD44v3-10 immunostaining in diagnostic evaluation of difficult serous effusions.

Nerve cell adhesion molecule expression in squamous cell carcinoma of the head and neck: a predictor of propensity toward perineural spread.

To evaluate head and neck squamous cell carcinomas (SCCAs) for the expression of nerve cell adhesion molecule (N-CAM). We propose that expression of N-CAM by tumor cells may be associated with perineural invasion in SCCA of the head and neck. Seventy-six archived specimens of histologically proven SCCA were analyzed by immunohistochemistry for the expression of N-CAM. Positive and negative controls were used to assess staining. Two sections of each specimen were reviewed for the presence of perineural invasion. A retrospective chart review was performed for each patient that corresponded to the above specimens. Perineural invasion was present in 28 (37%) of the 76 patients evaluated for the expression of N-CAM. N-CAM expression was demonstrated in 38 (50%) of the 76 specimens. The incidence of N-CAM expression was significantly associated with perineural invasion (P = .002). There was no significant association between the presence of staining or the presence of perineural invasion and the incidence of locoregional recurrence, distant metastasis, or survival status; however, the mean follow-up was only 13.6 months (range, 1-49 mo). There is a positive correlation between the presence of N-CAM expression and perineural invasion in SCCA of the head and neck. The expression of this adhesion molecule by tumor cells may facilitate both homophilic cell-to-cell and heterophilic cell-to-substrate adhesion, thereby enabling the tumor cells to use the perineural tissues or neural cells, or both as a conduit for perineural spread.

Fos‐like immunoreactivity in central auditory neurons of the mouse

Fos-like immunoreactivity was used to study sound-induced activation of neurons in the auditory brainstem. Immunoreactivity was assayed with a polyclonal antibody to Fos. In response to 6-kHz tone bursts, the pattern of staining was a band of immunoreactive neurons positioned at the tonotopically appropriate position within the cochlear nucleus and the inferior colliculus. The band was narrow at low sound pressure levels but wider along the tonotopic axis at higher sound levels. In response to noise bursts, the pattern was broader and often extended throughout the auditory nuclei. Often within this broad pattern were "sub-bands" of immunostained neurons, interspersed with bands of unstained neurons. With increasing sound pressure levels above 35-55 dB, the number of Fos-like immunoreactive neurons increased for the cochlear nucleus, superior olivary complex, and inferior colliculus. In the cochlear nucleus and inferior colliculus, the stained cells were small, and hence their activity would be difficult to sample in electrophysiological studies. In the medial nucleus of the trapezoid body, the stained neurons had larger somata and other characteristics of principal cells. Anesthesia with Nembutal or Avertin, but not with ketamine or urethane, decreased the number of Fos-like immunoreactive neurons in the cochlear nucleus. The different anesthetics produced more variable results in the inferior colliculus. In anesthetized, monaurally stimulated animals, the presence of staining in the contralateral cochlear nucleus indicates that some Fos-like immunoreactivity may be mediated by descending or commissural systems. These observations indicate that Fos assays are useful for studying the pattern of neuronal activation in the auditory system and may also be useful in studying the descending auditory pathways.

The late effects of radiation on the blood brain barrier

Rats were irradiated with 60 to 20 Gy in a single dose focussed to a volume of 0.5 cc in the center of the left hemisphere. Breakdown of the BBB was detected by (a) the presence of spikes on EEG after subcutaneous injection of Bicuculline methiodide, and (b) the presence of staining following Evans Blue dye injection. Breakdown of the BBB (mean and standard deviation) occurred in 38 46 of the 60 Gy rats at 98 ± 16 days, 7 11 of the 50 Gy rats at 128 ± 25 days, 11 18 of the 40 Gy rats at 162 ± 3 days, 5 11 of the 30 Gy rats at 178 ± 5 days and, 7 12 of the 20 Gy rats at 217 ± 7 days post irradiation. This study suggests that endothelial damage could be the principle mechanism mediating the late radiation syndrome.