INI1 (BAF 47) immunohistochemistry is an essential diagnostic tool for children with hepatic tumors and low alpha fetoprotein

Malignant rhabdoid tumor (MRT) of the liver is a rare malignancy with grave prognosis. This entity should be considered in the differential diagnosis of any aggressive liver tumor with low levels of alpha fetoprotein. We report 2 cases of hepatic MRT presenting in infancy. In these 2 cases, we show that loss of INI1 facilitates making the correct diagnosis of primary hepatic MRT utilizing BAF 47 (INI1 gene product) immunostains. Difficulty encountered in making this rare diagnosis, including the need for repeated biopsies, can be avoided if MRT is considered in the differential diagnosis early on and BAF 47 immunohistochemistry is ordered.

Conjunctival Proliferation After a Mild Pepper Spray Injury in a Young Child

PURPOSE To report a case of conjunctival proliferation in a 2.5-year-old boy after initial evidence of a mild chemical injury after ocular exposure to pepper spray (oleoresin capsicum). METHODS Case report with ophthalmologic and histologic findings. RESULTS A child presented with mild conjunctival injection and chemosis without any corneal erosion after direct exposure to pepper spray. Three weeks later, a significant conjunctival proliferation was found at the limbus, which was refractory to treatment with topical corticosteroids. Finally, proliferative tissue was surgically excised without clinical recurrence during 2 months of follow-up. CONCLUSIONS We hypothesize that the young age of the patient may have been an important factor for the severe conjunctival proliferation in comparison to a mainly uncomplicated course of pepper spray injuries in most adults. We recommend the use of topical antiinflammatory treatment even in apparently mild pepper spray injuries, especially in young children.

Characterization of antibodies specific for canine TLR4, 5 and 9 by ELISA, Western blotting and immunohistochemistry

Toll-like receptors recognize pathogen-associated molecular patterns of microbial origin, and ligand recognition results in the production of different immune mediators such as pro-inflammatory cytokines, interferon, reactive oxygen and nitrogen intermediates, and upregulation of costimmulatory molecules. As these receptors have a critical role in linking pathogen recognition to induction of inflammation and innate as well as adaptive immunity, there is tremendous interest in understanding how the tissue and cell-type expression of TLRs is regulated and its influence on the local innate immune response. While TLRs are well studied in humans and rodents, to date little is known about them in dogs. The purpose of this study was to develop canine specific antibodies against TLR2, 4, 5 and 9 that were used to measure relative expression of these TLRs in healthy and reactive canine mesenteric lymph nodes. All 8 rabbit sera (2 each for TLR2, 4, 5 and 9) were strongly positive in ELISA against the respective 2 peptides per TLR used for immunization. The purified antibodies selected specifically detected a protein band with an apparent size of approximately 70 kDa in lysates of canine PBMCs by Western blotting. Immunostaining was observed with purified antibodies against TLR4, 5 and 9, whereas for canine TLR2, staining was only observed with the unpurified antibodies. In the mesenteric lymph node of healthy dogs, the overall staining pattern was very similar for TLR4 and 5 with positive cells predominantly found in the internodular areas and lower part of the cortex. Compared to the TLR4 and 5, more cells stained positive for TLR9 especially in the lymphoid nodules. The reactive lymph nodes contained more TLR4 and 9 positive cells. Moreover, a shift of TLR-9 positive cells from the lymphoid follicles to the deep cortex and medullary cords was observed. Whereas TLR9 co-localized with CD79-positive areas, TLR4 and 5 antibodies stained cells primarily in the CD3-positive areas. All three TLR antibodies stained cells within the area that co-localized with lysozyme-positive cells. In conclusion, this study demonstrates that the antibodies generated against canine TLR 4, 5 and 9 identify the expression of these TLRs in formalin-fixed canine lymph nodes and demonstrate increased expression in reactive canine mesenteric lymph nodes.

How reliable is Ki-67 immunohistochemistry in grade 2 breast carcinomas? A QA study of the Swiss Working Group of Breast- and Gynecopathologists

Adjuvant chemotherapy decisions in breast cancer are increasingly based on the pathologist's assessment of tumor proliferation. The Swiss Working Group of Gyneco- and Breast Pathologists has surveyed inter- and intraobserver consistency of Ki-67-based proliferative fraction in breast carcinomas. Methods Five pathologists evaluated MIB-1-labeling index (LI) in ten breast carcinomas (G1, G2, G3) by counting and eyeballing. In the same way, 15 pathologists all over Switzerland then assessed MIB-1-LI on three G2 carcinomas, in self-selected or pre-defined areas of the tumors, comparing centrally immunostained slides with slides immunostained in the different laboratoires. To study intra-observer variability, the same tumors were re-examined 4 months later. Results The Kappa values for the first series of ten carcinomas of various degrees of differentiation showed good to very good agreement for MIB-1-LI (Kappa 0.56–0.72). However, we found very high inter-observer variabilities (Kappa 0.04–0.14) in the read-outs of the G2 carcinomas. It was not possible to explain the inconsistencies exclusively by any of the following factors: (i) pathologists' divergent definitions of what counts as a positive nucleus (ii) the mode of assessment (counting vs. eyeballing), (iii) immunostaining technique, and (iv) the selection of the tumor area in which to count. Despite intensive confrontation of all participating pathologists with the problem, inter-observer agreement did not improve when the same slides were re-examined 4 months later (Kappa 0.01–0.04) and intra-observer agreement was likewise poor (Kappa 0.00–0.35). Conclusion Assessment of mid-range Ki-67-LI suffers from high inter- and intra-observer variability. Oncologists should be aware of this caveat when using Ki-67-LI as a basis for treatment decisions in moderately differentiated breast carcinomas.

Somatostatin receptor subtype 2A immunohistochemistry using a new monoclonal antibody selects tumors suitable for in vivo somatostatin receptor targeting

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

Effects of daunorubicin, mitomycin C, azathioprine and cyclosporin A on human retinal pigmented epithelial, corneal endothelial and conjunctival cell lines

BACKGROUND: We wished to investigate the toxicity of four immunosuppressant and antimetabolic drugs, which are known to influence postoperative wound healing, on three different human ocular cell lines. METHODS: Acute toxicity to cyclosporin A, azathioprine, mitomicyn C and daunorubicin was assessed in Chang cells by monitoring their uptake of propidium iodide during a 3-h period. Chronic toxicity was assessed by monitoring the proliferation and viability of subconfluent cultures of Chang cells, human corneal endothelial cells (HCECs) and retinal pigmented epithelial (RPE) cells after continuous exposure to the drugs for 7 days. RESULTS: Acute toxicity testing revealed no obvious effects. However, the chronic toxicity tests disclosed a narrow concentration range over which cell proliferation decreased dramatically but calcein metabolism was sustained. Although the three lines reacted similarly to each agent, HCECs were the most vulnerable to daunorubicin and mitomycin. At a daunorubicin concentration of 0.05 microg/ml, a 75% decrease in calcein metabolism (P < 0.001) and a > or = 95% cell loss (P < 0.001) were observed. At a mitomycin concentration of 0.01 mug/ml, cell density decreased by 61% (P < 0.001) without a change in calcein metabolism, but at 0.1 microg/ml, the latter parameter decreased to 12% (P = 0.00014). At this concentration the proliferation of Chang and RPE cells decreased by more than 50%, whilst calcein metabolism was largely sustained. Cyclosporin inhibited cell proliferation moderately at lower concentrations (< 5 microg/ml; P=0.05) and substantially at higher ones, with a corresponding decline in calcein metabolism. Azathioprine induced a profound decrease in both parameters at concentrations above 5 microg/ml. CONCLUSION: Daunorubicin, cyclosporin and azathioprine could be used to inhibit excessive intraocular scarring after glaucoma and vitreoretinal surgery without overly reducing cell viability. The attributes of immunosuppressants lie in their combined antiproliferative and immunomodulatory effects.

Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Determination of the molecular subtypes of diffuse large B-cell lymphomas using immunohistochemistry: a case series from the Inselspital, Bern, and a critical appraisal of this determination in Switzerland

The two major subtypes of diffuse large B-cell lymphoma (DLBCL) (germinal centre B-cell - like (GCB-DLBCL) and activated B-cell - like (ABC-DLBCL)) are defined by means of gene expression profiling (GEP). Patients with GCB-DLBCL survive longer with the current standard regimen R-CHOP than patients with ABC-DLBCL. As GEP is not part of the current routine diagnostic work-up, efforts have been made to find a substitute than involves immunohistochemistry (IHC). Various algorithms achieved this with 80-90% accuracy. However, conflicting results on the appropriateness of IHC have been reported. Because it is likely that the molecular subtypes will play a role in future clinical practice, we assessed the determination of the molecular DLBCL subtypes by means of IHC at our University Hospital, and some aspects of this determination elsewhere in Switzerland. The most frequently used Hans algorithm includes three antibodies (against CD10, bcl-6 and MUM1). From records of the routine diagnostic work-up, we identified 51 of 172 (29.7%) newly diagnosed and treated DLBCL cases from 2005 until 2010 with an assigned DLBCL subtype. DLBCL subtype information was expanded by means of tissue microarray analysis. The outcome for patients with the GCB subtype was significantly better compared with those with the non-GC subtype, independent of the age-adjusted International Prognostic Index. We found a lack of standardisation in the subtype determination by means of IHC in Switzerland and significant problems of reproducibility. We conclude that the Hans algorithm performs well in our hands and that awareness of this important matter is increasing. However, outside clinical trials, vigorous efforts to standardise IHC determination are needed as DLBCL subtype-specific therapies emerge.