Immunostaining Patterns of Posttransplant Liver Biopsies Using 2 Anti-C4d Antibodies.

Histopathologic diagnosis of antibody-mediated rejection in posttransplant liver biopsies is challenging. The recently proposed diagnostic criteria by the Banff Working Group on Liver Allograft Pathology require positive C4d immunohistochemical staining to establish the diagnosis. However, the reported C4d staining patterns vary widely in different studies. One potential explanation may be due to different antibody preparations used by different investigators. In this study, posttransplant liver biopsies from 69 patients histopathologically diagnosed with acute cellular rejection, chronic rejection, or recurrent hepatitis C were immunohistochemically stained using 2 polyclonal anti-C4d antibodies. On the basis of the distribution of C4d immunoreactivity, 5 different staining patterns were observed: portal vein and capillary, hepatic artery, portal stroma, central vein, and sinusoids. The frequency, extent, and intensity of positive C4d staining with the 2 antibody preparations differed significantly for portal veins/capillaries and central veins, but not for hepatic arteries and portal stroma. Positive sinusoidal staining was seen in only 1 case. There were no significant differences in the frequency, extent, and intensity of positive C4d staining among the acute cellular rejection, chronic rejection, and recurrent hepatitis C groups with the 2 anti-C4d antibodies. These data show that different anti-C4d antibodies can show different staining patterns, which may lead to different interpretation. Caution is thus needed when selecting C4d antibodies for clinical use to aid in the diagnosis of antibody-mediated rejection.

Diagnostic value of maspin in distinguishing adenocarcinoma from benign biliary epithelium on endoscopic bile duct biopsy.

Histopathologic distinction between benign and malignant epithelia on endoscopic bile duct biopsy can be extremely challenging due to small sample size, crush artifact, and a propensity for marked inflammatory and reactive changes after stent placement. Our previous studies have shown that the insulin-like growth factor II mRNA-binding protein 3, S100P, and the von Hippel-Lindau gene product (pVHL) can help the distinction. This study analyzed 134 endoscopic bile duct biopsy specimens (adenocarcinoma 45, atypical 31, and benign 58) by immunohistochemistry for the expression of maspin, a serine protease inhibitor. The results demonstrated that (1) maspin expression was more frequently detected in malignant than in benign biopsies; (2) malignant biopsies frequently showed diffuse, strong/intermediate, and combined nuclear/cytoplasmic staining patterns for maspin, which were much less commonly seen in benign biopsies; (3) the malignant staining patterns for maspin observed in atypical biopsies were consistent with follow-up data showing that 67% of these patients were subsequently diagnosed with adenocarcinoma; (4) a maspin+/S100P+/pVHL- staining profile was seen in 75% of malignant biopsies but in none of the benign cases. These observations demonstrate that maspin is a useful addition to the diagnostic immunohistochemical panel (S100P, pVHL, and insulin-like growth factor II mRNA-binding protein 3) to help distinguish malignant from benign epithelia on challenging bile duct biopsies.

Immunohistochemical stains of proliferating cell nuclear antigen, insulin-like growth factor 2 and clusterin help distinguish malignant from benign liver nodular lesions.

AIMS: To explore the immunohistochemical utility of proliferating cell nuclear antigen (PCNA), insulin-like growth factor 2 (IGF2) and clusterin in the distinction between malignant and benign liver nodular lesions. METHODS: Immunohistochemical stains for PCNA, IGF2 and clusterin were performed on 284 liver nodular lesions, including 33 hepatocellular adenomas (HCA), 40 focal nodular hyperplasias (FNH), 77 large regenerative nodules (LRN) and 134 hepatocellular carcinomas (HCC). RESULTS: Strong and diffuse nuclear PCNA immunoreactivity was observed in 103 (77%) HCCs but in only 2 (6%) HCAs. None of the FNH and LRN cases showed a strong and diffuse staining pattern. All HCAs, 95% of FNHs and 92% of LRNs showed cytoplasmic IGF2 expression, with a strong staining observed in 70% of HCAs, 20% of FNHs and 30% of LRNs. This was in marked contrast to that observed in HCCs, where 66% of HCCs demonstrated a weak and focal/patchy immunostaining pattern and another 25% showed no detectable IGF2 immunoreactivity. In comparison with their adjacent non-lesional hepatocytes, 75% of HCCs showed decreased IGF2 expression. However, decreased IGF2 expression was not evident in HCAs, FNHs and LRNs. Cytoplasmic staining for clusterin was seen in both benign and malignant nodular lesions. However, an enhanced and exaggerated pericanalicular staining pattern was observed in 75% of HCCs, which was not demonstrated in HCAs, FNHs and LRNs. CONCLUSIONS: PCNA, IGF2 and clusterin show unique immunostaining characteristics in HCCs, which can be useful adjuncts to other currently available markers to aid in the distinction of HCC from benign liver nodular lesions.

Use of IMP3, S100P, and pVHL immunopanel to aid in the interpretation of bile duct biopsies with atypical histology or suspicious for malignancy.

Histologic evaluation of an endoscopic bile duct biopsy for malignancy is a known challenge. Our prior study has shown that the insulin-like growth factor-II mRNA binding protein-3 (IMP3), S100P, and the von Hippel-Lindau gene product (pVHL) are a useful immunopanel for the distinction between adenocarcinoma and benign biliary epithelium. To further evaluate the usefulness of the IMP3, S100P, and pVHL immunopanel to aid in the interpretation of bile duct biopsies, 16 histologically challenging bile duct biopsies that exhibited atypical histology or features suspicious for malignancy were immunohistochemically stained for IMP3, S100P, and pVHL. Clinical follow-up data were obtained for each case. The results showed that in the 11 cases that showed adenocarcinoma during follow-up, the following staining patterns in atypical/suspicious cells in the initial biopsies were observed: IMP3-positive/S100P-positive/pVHL-negative or reduced (n=6), IMP3-negative/S100P-positive/pVHL-negative or reduced (n=4), and IMP3-positive/S100P-negative/pVHL-negative (n=1). In the 5 follow-up-proven benign cases, 2 biopsies showed an IMP3-negative/S100P-positive/pVHL-positive pattern in atypical cells and 1 was negative for all 3 proteins. The remaining 2 biopsies exhibited an IMP3-positive/S100P-positive/pVHL-negative or reduced pattern in atypical cells that were histologically considered dysplastic on retrospective review. These observations reaffirm that bile duct adenocarcinoma frequently shows positive IMP3 and/or S100P staining with reciprocal negative or reduced pVHL expression. This staining pattern can also be seen in dysplastic epithelium in the absence of invasive carcinoma. On the contrary, benign biliary epithelium typically lacks IMP3 immunoreactivity and may retain normal pVHL expression. However, caution should be exercised when using this immunopanel in the interpretation of challenging bile duct biopsies, because S100P and pVHL stains may give rise to variable patterns that can be difficult to interpret.

Diagnostic utility of CD10 in benign and malignant extrahepatic bile duct lesions.

CD10, a cell surface enzyme with neutral metalloendopeptidase activity, is a marker for intestinal epithelial brush border. It is also present in normal bile ducts and gallbladder epithelia but is absent in cholangiocarcinomas. However, the expression profile of CD10 in benign and malignant extrahepatic biliary lesions has not been studied. In this study, 69 biopsies, 9 resections, and 9 cell blocks prepared from fine-needle aspirations of the extrahepatic bile ducts from 86 patients were studied immunohistochemically for CD10 expression. The majority of cases contained normal biliary epithelium (NL, n=64), along with foci of benign or malignant lesions in various combinations. Benign lesions included reactive atypia (n=35), low-grade dysplasia of unknown significance (n=21), and bile duct adenoma (BDA, n=1). Malignant lesions included high-grade dysplasia (HGD, n=45) and invasive adenocarcinoma (IC, n=30). As expected, the NL showed strong continuous staining at the apical surface in all cases. Benign lesions were also CD10 positive in all but 3 cases; however, the staining pattern was discontinuous, with positive cells varying from 20% to 80%. None of the malignant lesions showed CD10 immunoreactivity, except for 2 HGD cases and 1 IC case, which exhibited focal staining. The Pearson χ2 and Fisher exact tests showed significant statistical difference in CD10 expression among the study groups (P<0.001). Our findings suggest that absence of CD10 expression in strips of atypical biliary epithelial cells may be a phenotype associated with malignant transformation and may serve as a useful marker to aid in the evaluation of bile duct biopsies, in which distinction between benign and malignant lesions on biopsies or cytology specimens can be extremely challenging because of limited sampling, crush artifact, and frequent inflammatory/reactive changes.

Injection site pseudosarcoma in piriformis syndrome.

AIMS: Pseudosarcomatous reactive myofibroblastic proliferations have been described following surgery or trauma at a variety of anatomical sites. These types of reactions have not been previously described at injection sites. Here we evaluated prevalence, morphologic patterns and clinical resolution of such lesions. METHODS AND RESULTS: We analyzed 266 surgical resection specimens obtained during the definitive treatment of piriformis syndrome. Three cases showed exuberant reactive fibroblastic/myofibroblastic intramuscular proliferations, mimicking a sarcoma. In all three cases the surgeries were found to be preceded by local injections of cortisone and bupivacaine. Clinical follow-up revealed no uncontrolled growth. CONCLUSIONS: As the clinical history of injections is often not provided, it is important to be aware of this pitfall when reviewing skeletal muscle resections for entrapment syndromes.

Lack of HER2 overexpression and amplification in small intestinal adenocarcinoma.

HER2 overexpression and amplification have been studied as a therapeutic and prognostic target in a number of human cancers, including esophageal, gastric, and colorectal adenocarcinomas. However, HER2 status has not been well investigated in primary small intestinal adenocarcinoma, probably because of its rarity. In this study, we conducted immunohistochemical analysis and fluorescence in situ hybridization (FISH) for HER2 on 49 primary nonampullar small intestinal adenocarcinomas. The results showed a complete lack of HER2 protein expression in 47 cases (96%) by immunohistochemical analysis. Only 2 cases (4%) showed a 1+ staining pattern. No tumors exhibited 2+ or 3+ HER2 immunoreactivity. By FISH, none of the tumors, including those with 1+ HER2 immunoreactivity, exhibited HER2 gene amplification. These observations demonstrate that HER2 protein overexpression and gene amplification are infrequent events, if they occur at all, in small intestinal adenocarcinoma. Thus, routine immunohistochemical and/or FISH testing for HER2 for potential targeted anti-HER2 therapy may not be beneficial for patients with primary small intestinal adenocarcinoma.

Characterization of three Staphylococcus aureus isolates from a 17-year-old female who died of tampon-related toxic shock syndrome.

We report the identification and characterization of three Staphylococcus aureus isolates recovered from throat and vaginal cultures, as well as from an axillary abscess, of a 17-year-old female who died of tampon-related toxic shock syndrome. The three S. aureus isolates were unrelated as determined by pulsed-field gel electrophoresis. The vaginal isolate was mecA, Panton-Valentine leukocidin, and staphylococcal enterotoxin B and C negative, toxic shock syndrome toxin 1 positive, and staphylococcal cassette chromosome mec element (SCCmec) untypeable, which was consistent with the clinical and autopsy findings that death was due to tampon-related toxic shock syndrome.

A specific antagonist of the p110delta catalytic component of phosphatidylinositol 3'-kinase, IC486068, enhances radiation-induced tumor vascular destruction.

The phosphatidylinositol 3'-kinase (PI3k)/protein kinase B (PKB/Akt) signal transduction pathway plays a critical role in mediating endothelial cell survival and function during oxidative stress. The role of the PI3k/Akt signaling pathway in promoting cell viability was studied in vascular endothelial cells treated with ionizing radiation. Western blot analysis showed that Akt was rapidly phosphorylated in response to radiation in primary culture endothelial cells (human umbilical vascular endothelial cells) in the absence of serum or growth factors. PI3k consists of p85 and p110 subunits, which play a central upstream role in Akt activation in response to exogenous stimuli. The delta isoform of the p110 subunit is expressed in endothelial cells. We studied the effects of the p110delta specific inhibitor IC486068, which abrogated radiation-induced phosphorylation of Akt. IC486068 enhanced radiation-induced apoptosis in endothelial cells and reduced cell migration and tubule formation of endothelial cells in Matrigel following irradiation. In vivo tumor growth delay was studied in mice with Lewis lung carcinoma and GL261 hind limb tumors. Mice were treated with daily i.p. injections (25 mg/kg) of IC486068 during 6 days of radiation treatment (18 Gy). Combined treatment with IC486068 and radiation significantly reduced tumor volume as compared with either treatment alone. Reduction in vasculature was confirmed using the dorsal skinfold vascular window model. The vascular length density was measured by use of the tumor vascular window model and showed IC486068 significantly enhanced radiation-induced destruction of tumor vasculature as compared with either treatment alone. IC486068 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. These findings suggest that p110delta is a therapeutic target to enhance radiation-induced tumor control.

SU11248 maintenance therapy prevents tumor regrowth after fractionated irradiation of murine tumor models.

Receptor tyrosine kinase activation contributes to cell viability during cytotoxic therapy. The novel broad spectrum receptor tyrosine kinase inhibitor, SU11248, inhibits vascular endothelial growth factor receptor 2, platelet-derived growth factor receptor, c-kit, and fetal liver tyrosine kinase 3. In this study, we maintained SU11248 plasma levels beyond the completion of radiotherapy to determine whether tumor regrowth can be delayed. The antiangiogenic effects of SU11248 were demonstrated using human umbilical vein endothelial cells in vitro. Apoptosis increased and clonogenic survival decreased when SU11248 was used in combination with radiation from 0 to 6 Gy on endothelial cells. In vivo tumor growth delay was increased in C57B6J mice with Lewis lung carcinoma or glioblastoma multiform (GL261) hind limb tumors. Mice were treated with daily i.p. injections (40 mg/kg) of SU11248 during 7 days of radiation treatment (21 Gy). Combined treatment with SU11248 and radiation significantly reduced tumor volume as compared with either treatment alone. Concomitant reduction in vasculature was confirmed using the dorsal vascular window model. The vascular length established using images taken from a consistent quadrant in the window show the combination therapy was more effective in destroying tumor vasculature than either treatment alone. SU11248 maintenance administration beyond the completion of radiotherapy results in prolongation of tumor control. In summary, SU11248 enhances radiation-induced endothelial cytotoxicity, resulting in tumor vascular destruction and tumor control when combined with fractionated radiotherapy in murine tumor models. Moreover, inhibition of angiogenesis well beyond radiation therapy may be a promising treatment paradigm for refractory human neoplasms.