Audrey N. Schuetz

Molecular classification of renal tumors by gene expression profiling.

Renal tumor classification is important because histopathological subtypes are associated with distinct clinical behavior. However, diagnosis is difficult because tumor subtypes have overlapping microscopic characteristics. Therefore, ancillary methods are needed to optimize classification. We used oligonucleotide microarrays to analyze 31 adult renal tumors, including clear cell renal cell carcinoma (RCC), papillary RCC, chromophobe RCC, oncocytoma, and angiomyolipoma. Expression profiles correlated with histopathology; unsupervised algorithms clustered 30 of 31 tumors according to appropriate diagnostic subtypes while supervised analyses identified significant, subtype-specific expression markers. Clear cell RCC overexpressed proximal nephron, angiogenic, and immune response genes, chromophobe RCC oncocytoma overexpressed distal nephron and oxidative phosphorylation genes, papillary RCC overexpressed serine protease inhibitors, and extracellular matrix products, and angiomyolipoma overexpressed muscle developmental, lipid biosynthetic, melanocytic, and distinct angiogenic factors. Quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry of formalin-fixed renal tumors confirmed overexpression of proximal nephron markers (megalin/low-density lipoprotein-related protein 2, alpha-methylacyl CoA racemase) in clear cell and papillary RCC and distal nephron markers (beta-defensin 1, claudin 7) in chromophobe RCC/oncocytoma. In summary, renal tumor subtypes were classified by distinct gene expression profiles, illustrating tumor pathobiology and translating into novel molecular bioassays using fixed tissue.

Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes

ABSTRACT We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of blaKPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the blaKPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the blaKPC PCR assay was used to confirm the presence of blaKPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected blaKPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of blaKPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two blaKPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with blaKPC-2 and blaKPC-3, respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.

Intercellular junctions in Ewing sarcoma/ primitive neuroectodermal tumor : additional evidence of epithelial differentiation

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) has recently been shown to frequently express cytokeratins, suggesting partial epithelial differentiation. Older ultrastructural studies have documented primitive cell–cell junctions in ES/PNET, reportedly resembling poorly formed desmosomes. Recently, paraffin-reactive antibodies have become available to proteins found in a variety of intercellular junctions indicative of epithelial differentiation, including tight junctions, desmosomes and adherens junctions. We examined intercellular junction protein expression in a large number of genetically confirmed ES/PNET. Formalin-fixed, paraffin-embedded sections from 23 primary and seven recurrent or metastatic cases of genetically confirmed ES/PNET were immunostained for claudin-1 and occludin (tight junction structural proteins), zonula occludens-1 (ZO-1, tight junction linker protein), desmoglein 1/2 (desmosomal adherens protein), desmoplakin (desmosomal structural protein) and E-cadherin (epithelial adherens junction protein), using steam heat-induced epitope retrieval and the Dako Envision system. Cases with >5% positive cells were scored as ‘positive’. Normal colonic epithelium and skin served as external positive controls. Claudin-1 was expressed by 19 of 30 specimens (63%), ZO-1 was expressed by 15 of 29 specimens (51%), and occludin was expressed by three of 28 specimens (11%). In 28 specimens all three tight junction markers were evaluable. In all, 15 samples (54%) expressed only one tight junction marker, and 10 samples (36%) expressed two tight junction markers. No case expressed all three tight junction markers. Desmoglein was expressed in one of 30 (3%) samples. Desmoplakin was expressed in two of 28 (7%) samples. E-cadherin was negative in all cases. Our data suggest that many of the previously described cell–cell junctions in ES/PNET are poorly formed tight junctions, given the high frequency of claudin-1 and ZO-1 expression. This may underestimate the true frequency of tight junction protein expression in ES/PNET, as there are at least 20 different claudins and other ZO proteins. These tight junctions are almost certainly abnormal, given the absence of occludin expression in most cases. Desmosomal and adherens junction protein expression was rare to absent. Our findings provide additional evidence that ES/PNET frequently show partial epithelial differentiation.

Aspergillus immunohistochemistry of culture-proven fungal tissue isolates shows high cross-reactivity.

Increased use of immunosuppressive drugs has broadened the range and increased the incidence of invasive fungal diseases. Successful therapy relies on early diagnosis; however, fungal organisms show overlapping morphology on histologic sections. As commercially available fungal immunohistochemistry (IHC) has been validated for crossreactivity with few other fungi, we ran Aspergillus and Candida IHCs on formalin-fixed, paraffin-embedded tissue of fungal cases. The cases had also been cultured from fresh tissue submitted to the clinical microbiology laboratory. Commercially available polyclonal and monoclonal Aspergillus and Candida IHCs were run with antigen retrieval on 16 culture-confirmed fungal cases (9 Aspergillus, 2 Candida, 2 Bipolaris, 1 Fusarium, 1 Curvularia, and 1 Rhizopus). Three additional cases, which stained positive for Zygomycete IHC at the Centers for Disease Control and Prevention, which had not been confirmed by culture, were also tested against the antibodies. Preabsorption controls with formalin-fixed fungi were run on 2 cases. Fifteen of the 16 culture-confirmed cases stained with the polyclonal Aspergillus antibody, whereas only 7 of 14 cases stained with the monoclonal Aspergillus antibody. Sensitivities and specificities for the polyclonal and monoclonal Aspergillus antibodies were 100% and 29%, and 43% and 14%, respectively. Two Zygomycete IHC Centers for Disease Control and Prevention cases stained strongly positive for monoclonal Aspergillus. Although Aspergillus cultures were often correctly identified on IHC, false positive staining was seen with both polyclonal and monoclonal Aspergillus antibodies. The number of culture-confirmed Candida cases was too low to adequately evaluate sensitivity. More accurate and reliable fungal IHCs, including those for Zygomycete staining, are needed.

Infectious Disease Immunohistochemistry in Placentas from HIV-Positive and HIV-Negative Patients

Studies comparing placental pathology between human immunodeficiency virus (HIV)–positive and HIV-negative patients have shown conflicting results. In addition, few studies have evaluated the infectious etiology of placental inflammation in HIV-positive patients. We examined a cohort of placentas from 73 HIV-positive and 41 HIV-negative patients to gain a better understanding of the spectrum of placental inflammatory lesions. Bacterial and viral immunohistochemistry (IHC) was run on a subset of placentas (12 HIV-positive and 7 HIV-negative) with the greatest amount of inflammation. Although few histologic differences were seen between the HIV-positive and HIV-negative groups, chorioamnionitis was of a higher stage in the HIV-positive placentas. An infectious agent was found by IHC in 3 of 7 HIV-negative patients (2 Neisseria spp. and 1 group B Streptococcus). One HIV-positive placenta showed gram-positive cocci on fetal membranes; organisms were not detected by IHC. In 2 patients, the etiologic agent was not suspected prior to IHC. This study identified that acute inflammation is less common in placentas from HIV-positive patients, compared with HIV-negative patients. However, when severe inflammation is present, infectious organisms may be identified by IHC, providing a more specific diagnosis and offering a beneficial impact in maternal and fetal management.

Towards the prevention of transfusion-transmitted infectious diseases

Transfusion-transmission of viral infections, such as HIV and hepatitis C virus, were once the scourge of blood transfusion. However, due to remarkable progress over the last 30 years, tests for viral proteins, antibody responses and more recently, viral nucleic acids, have virtually eliminated these risks. This review summarizes these advances in an historical context, describes new methodologies on the horizon, and discusses residual infectious risks associated with blood transfusion.